SRRF: Universal live-cell super-resolution microscopy.

PubMed

Culley, Siân; Tosheva, Kalina L; Matos Pereira, Pedro; Henriques, Ricardo

2018-08-01

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

NDE of structural ceramics

NASA Technical Reports Server (NTRS)

Klima, S. J.; Vary, A.

1986-01-01

Radiographic, ultrasonic, scanning laser acoustic microscopy (SLAM), and thermo-acoustic microscopy techniques were used to characterize silicon nitride and silicon carbide modulus-of-rupture test specimens in various stages of fabrication. Conventional and microfocus X-ray techniques were found capable of detecting minute high density inclusions in as-received powders, green compacts, and fully densified specimens. Significant density gradients in sintered bars were observed by radiography, ultrasonic velocity, and SLAM. Ultrasonic attenuation was found sensitive to microstructural variations due to grain and void morphology and distribution. SLAM was also capable of detecting voids, inclusions and cracks in finished test bars. Consideration is given to the potential for applying thermo-acoustic microscopy techniques to green and densified ceramics. The detection probability statistics and some limitations of radiography and SLAM also are discussed.

Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

PubMed Central

Delaney, P M; King, R G; Lambert, J R; Harris, M R

1994-01-01

Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

Three dimensional electron microscopy and in silico tools for macromolecular structure determination

PubMed Central

Borkotoky, Subhomoi; Meena, Chetan Kumar; Khan, Mohammad Wahab; Murali, Ayaluru

2013-01-01

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it. PMID:27092033

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device.

PubMed

Park, Jong Kang; Rowlands, Christopher J; So, Peter T C

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

PubMed Central

Park, Jong Kang; Rowlands, Christopher J.; So, Peter T. C.

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice. PMID:29387484

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Superresolution microscopy for microbiology

PubMed Central

Coltharp, Carla; Xiao, Jie

2014-01-01

Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

NASA Astrophysics Data System (ADS)

Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C.

2011-03-01

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levin, Barnaby D. A.; Zachman, Michael J.; Werner, Jörg G.

Abstract Lithium sulfur (Li–S) batteries have the potential to provide higher energy storage density at lower cost than conventional lithium ion batteries. A key challenge for Li–S batteries is the loss of sulfur to the electrolyte during cycling. This loss can be mitigated by sequestering the sulfur in nanostructured carbon–sulfur composites. The nanoscale characterization of the sulfur distribution within these complex nanostructured electrodes is normally performed by electron microscopy, but sulfur sublimates and redistributes in the high-vacuum conditions of conventional electron microscopes. The resulting sublimation artifacts render characterization of sulfur in conventional electron microscopes problematic and unreliable. Here, we demonstratemore » two techniques, cryogenic transmission electron microscopy (cryo-TEM) and scanning electron microscopy in air (airSEM), that enable the reliable characterization of sulfur across multiple length scales by suppressing sulfur sublimation. We use cryo-TEM and airSEM to examine carbon–sulfur composites synthesized for use as Li–S battery cathodes, noting several cases where the commonly employed sulfur melt infusion method is highly inefficient at infiltrating sulfur into porous carbon hosts.« less

Comparison of five methods of malaria detection in the outpatient setting.

PubMed

Lema, O E; Carter, J Y; Nagelkerke, N; Wangai, M W; Kitenge, P; Gikunda, S M; Arube, P A; Munafu, C G; Materu, S F; Adhiambo, C A; Mukunza, H K

1999-02-01

In eastern Africa where 90% of the malaria is due to Plasmodium falciparum, the accuracy of malaria diagnosis at the outpatient level is becoming increasingly important due to problems of drug resistance and use of alternative, costly antimalarial drugs. The quantitative buffy coat (QBC) technique, acridine orange staining with an interference filter system, and the ParaSight-F test have been introduced as alternative methods to conventional microscopy for the diagnosis of malaria. Two hundred thirteen outpatients were tested using these alternative methods and conventional microscopy by five experienced technologists; two were randomly allocated to read the results of each test. Paired results showed the highest level of agreement with the ParaSight-F test (99%), followed by Field stain (92%). The results of the QBC technique showed the least agreement (73%). Using conventional microscopy as the reference standard, the ParaSight-F test had a sensitivity range of 90-92% and a specificity of 99%, staining with acridine orange had a sensitivity range of 77-96% and a specificity range of 81-98% and the QBC technique had a sensitivity range of 88-98% and a specificity range of 58-90%. All microscopic tests showed lower sensitivities (as low as 20% using staining with acridine orange) in detecting low parasitemias (< or = 320/microl) than the ParaSight-F test (70%). Due to the high cost of the ParaSight-F test, Field-stained blood films remain the most appropriate method for diagnosis of P. falciparum in eastern Africa. The ParaSight-F test may be used in situations where no trained microscopists are available, or where malaria is strongly suspected and the results of microscopy are negative.

Single particle analysis based on Zernike phase contrast transmission electron microscopy.

PubMed

Danev, Radostin; Nagayama, Kuniaki

2008-02-01

We present the first application of Zernike phase-contrast transmission electron microscopy to single-particle 3D reconstruction of a protein, using GroEL chaperonin as the test specimen. We evaluated the performance of the technique by comparing 3D models derived from Zernike phase contrast imaging, with models from conventional underfocus phase contrast imaging. The same resolution, about 12A, was achieved by both imaging methods. The reconstruction based on Zernike phase contrast data required about 30% fewer particles. The advantages and prospects of each technique are discussed.

Tomographic diffractive microscopy with agile illuminations for imaging targets in a noisy background.

PubMed

Zhang, T; Godavarthi, C; Chaumet, P C; Maire, G; Giovannini, H; Talneau, A; Prada, C; Sentenac, A; Belkebir, K

2015-02-15

Tomographic diffractive microscopy is a marker-free optical digital imaging technique in which three-dimensional samples are reconstructed from a set of holograms recorded under different angles of incidence. We show experimentally that, by processing the holograms with singular value decomposition, it is possible to image objects in a noisy background that are invisible with classical wide-field microscopy and conventional tomographic reconstruction procedure. The targets can be further characterized with a selective quantitative inversion.

Nanoscale surface characterization using laser interference microscopy

NASA Astrophysics Data System (ADS)

Ignatyev, Pavel S.; Skrynnik, Andrey A.; Melnik, Yury A.

2018-03-01

Nanoscale surface characterization is one of the most significant parts of modern materials development and application. The modern microscopes are expensive and complicated tools, and its use for industrial tasks is limited due to laborious sample preparation, measurement procedures, and low operation speed. The laser modulation interference microscopy method (MIM) for real-time quantitative and qualitative analysis of glass, metals, ceramics, and various coatings has a spatial resolution of 0.1 nm for vertical and up to 100 nm for lateral. It is proposed as an alternative to traditional scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods. It is demonstrated that in the cases of roughness metrology for super smooth (Ra >1 nm) surfaces the application of a laser interference microscopy techniques is more optimal than conventional SEM and AFM. The comparison of semiconductor test structure for lateral dimensions measurements obtained with SEM and AFM and white light interferometer also demonstrates the advantages of MIM technique.

Tomographic phase microscopy and its biological applications

NASA Astrophysics Data System (ADS)

Choi, Wonshik

2012-12-01

Conventional interferometric microscopy techniques such as digital holographic microscopy and quantitative phase microscopy are often classified as 3D imaging techniques because a recorded complex field image can be numerically propagated to a different depth. In a strict sense, however, a single complex field image contains only 2D information on a specimen. The measured 2D image is only a subset of the 3D structure. For the 3D mapping of an object, multiple independent 2D images are to be taken, for example at multiple incident angles or wavelengths, and then combined by the so-called optical diffraction tomography (ODT). In this Letter, tomographic phase microscopy (TPM) is reviewed that experimentally realizes the concept of the ODT for the 3D mapping of biological cells in their native state, and some of its interesting biological and biomedical applications are introduced. [Figure not available: see fulltext.

Hall versus conventional stainless steel crown techniques: in vitro investigation of marginal fit and microleakage using three different luting agents.

PubMed

Erdemci, Zeynep Yalçınkaya; Cehreli, S Burçak; Tirali, R Ebru

2014-01-01

This study's purpose was to investigate microleakage and marginal discrepancies in stainless steel crowns (SSCs) placed using conventional and Hall techniques and cemented with three different luting agents. Seventy-eight human primary maxillary second molars were randomly assigned to two groups (N=39), and SSCs were applied either with the Hall or conventional technique. These two groups were further subgrouped according to the material used for crown cementation (N=13 per group). Two specimens in each group were processed for scanning electron microscopy investigation. The extent of microleakage and marginal fit was quantified in millimeters on digitally photographed sections using image analysis software. The data were compared with a two-way independent and a two-way mixed analysis of variance (P=.05). The scores in the Hall group were significantly worse than those in the conventional technique group (P<.05). In both groups, resin cement displayed the lowest extent of microleakage, followed by glass ionomer and polycarboxylate cements (P<.05). Stainless steel crowns applied using the Hall technique displayed higher microleakage scores than those applied using the conventional technique, regardless of the cementation material. When the interaction of the material and technique was assessed, resin cement presented as the best choice for minimizing microleakage in both techniques.

Label-free nanoscale characterization of red blood cell structure and dynamics using single-shot transport of intensity equation

NASA Astrophysics Data System (ADS)

Poola, Praveen Kumar; John, Renu

2017-10-01

We report the results of characterization of red blood cell (RBC) structure and its dynamics with nanometric sensitivity using transport of intensity equation microscopy (TIEM). Conventional transport of intensity technique requires three intensity images and hence is not suitable for studying real-time dynamics of live biological samples. However, assuming the sample to be homogeneous, phase retrieval using transport of intensity equation has been demonstrated with single defocused measurement with x-rays. We adopt this technique for quantitative phase light microscopy of homogenous cells like RBCs. The main merits of this technique are its simplicity, cost-effectiveness, and ease of implementation on a conventional microscope. The phase information can be easily merged with regular bright-field and fluorescence images to provide multidimensional (three-dimensional spatial and temporal) information without any extra complexity in the setup. The phase measurement from the TIEM has been characterized using polymeric microbeads and the noise stability of the system has been analyzed. We explore the structure and real-time dynamics of RBCs and the subdomain membrane fluctuations using this technique.

Customized atomic force microscopy probe by focused-ion-beam-assisted tip transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Andrew; Butte, Manish J., E-mail: manish.butte@stanford.edu

2014-08-04

We present a technique for transferring separately fabricated tips onto tipless atomic force microscopy (AFM) cantilevers, performed using focused ion beam-assisted nanomanipulation. This method addresses the need in scanning probe microscopy for certain tip geometries that cannot be achieved by conventional lithography. For example, in probing complex layered materials or tall biological cells using AFM, a tall tip with a high-aspect-ratio is required to avoid artifacts caused by collisions of the tip's sides with the material being probed. We show experimentally that tall (18 μm) cantilever tips fabricated by this approach reduce squeeze-film damping, which fits predictions from hydrodynamic theory, andmore » results in an increased quality factor (Q) of the fundamental flexural mode. We demonstrate that a customized tip's well-defined geometry, tall tip height, and aspect ratio enable improved measurement of elastic moduli by allowing access to low-laying portions of tall cells (T lymphocytes). This technique can be generally used to attach tips to any micromechanical device when conventional lithography of tips cannot be accomplished.« less

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

[Experimental research on the effect of nanophase ceramics on osteoblasts functions].

PubMed

Wen, Bo; Chen, Zhiqing; Jiang, Yinshan; Yang, Zhengwen; Xu, Yongzhong

2005-06-01

In order to study the cytocompatibility of nanophase hydroxyapatite ceramic in vitro, we prepared hydroxyapatite by use of the wet chemistry techniques. The grain size of hydroxyapatite of interest to the present study was determined by scanning electron microscopy and atomic force microscopy with image analysis software. Primary culture of osteoblast from rat calvaria was established. Protein content, synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase hydroxyapatite ceramics and on conventional hydroxyapatite ceramics for 7, 14, 21 and 28 days were examined. The results showed that the average surface grain size of the nanophase and that of the conventional HA compact formulations was 55 (nanophase) and 780 (conventional) nm, respectively. More importantly, compared to the synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase was significantly greater than that on conventional ceramics after 21 and 28 days. The cytocompatibility was significantly greater on nanophase HA than on conventional formulations of the same ceramic.

Imaging slit-coupled surface plasmon polaritons using conventional optical microscopy.

PubMed

Mehfuz, R; Chowdhury, F A; Chau, K J

2012-05-07

We develop a technique that now enables surface plasmon polaritons (SPPs) coupled by nano-patterned slits in a metal film to be detected using conventional optical microscopy with standard objective lenses. The crux of this method is an ultra-thin polymer layer on the metal surface, whose thickness can be varied over a nanoscale range to enable controllable tuning of the SPP momentum. At an optimal layer thickness for which the SPP momentum matches the momentum of light emerging from the slit, the SPP coupling efficiency is enhanced about six times relative to that without the layer. The enhanced efficiency results in distinctive and bright plasmonic signatures near the slit visible by naked eye under an optical microscope. We demonstrate how this capability can be used for parallel measurement through a simple experiment in which the SPP propagation distance is extracted from a single microscope image of an illuminated array of nano-patterned slits on a metal surface. We also use optical microscopy to image the focal region of a plasmonic lens and obtain results consistent with a previously-reported results using near-field optical microscopy. Measurement of SPPs near a nano-slit using conventional and widely-available optical microscopy is an important step towards making nano-plasmonic device technology highly accessible and easy-to-use.

Speckle-field digital holographic microscopy.

PubMed

Park, YongKeun; Choi, Wonshik; Yaqoob, Zahid; Dasari, Ramachandra; Badizadegan, Kamran; Feld, Michael S

2009-07-20

The use of coherent light in conventional holographic phase microscopy (HPM) poses three major drawbacks: poor spatial resolution, weak depth sectioning, and fixed pattern noise due to unwanted diffraction. Here, we report a technique which can overcome these drawbacks, but maintains the advantage of phase microscopy - high contrast live cell imaging and 3D imaging. A speckle beam of a complex spatial pattern is used for illumination to reduce fixed pattern noise and to improve optical sectioning capability. By recording of the electric field of speckle, we demonstrate high contrast 3D live cell imaging without the need for axial scanning - neither objective lens nor sample stage. This technique has great potential in studying biological samples with improved sensitivity, resolution and optical sectioning capability.

Detection of living Sarcoptes scabiei larvae by reflectance mode confocal microscopy in the skin of a patient with crusted scabies

NASA Astrophysics Data System (ADS)

Levi, Assi; Mumcuoglu, Kosta Y.; Ingber, Arieh; Enk, Claes D.

2012-06-01

Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

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.

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

PubMed

Okumura, Taiga; Shoji, Minami; Hisada, Akiko; Ominami, Yusuke; Ito, Sukehiro; Ushiki, Tatsuo; Nakajima, Masato; Ohshima, Takashi

2018-01-01

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

Handheld Fluorescence Microscopy based Flow Analyzer.

PubMed

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Confocal laser scanning microscopy of porcine skin: implications for human wound healing studies

PubMed Central

VARDAXIS, N. J.; BRANS, T. A.; BOON, M. E.; KREIS, R. W.; MARRES, L. M.

1997-01-01

The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model. PMID:9183682

Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

PubMed

Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan

2013-05-01

Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

A study of reduced chromium content in a nickel-base superalloy via element substitution and rapid solidification processing. Ph.D. ThesisFinal Report

NASA Technical Reports Server (NTRS)

Powers, William O.

1987-01-01

A study of reduced chromium content in a nickel base superalloy via element substitution and rapid solidification processing was performed. The two elements used as partial substitutes for chromium were Si and Zr. The microstructure of conventionally solidified materials was characterized using microscopy techniques. These alloys were rapidly solidified using the chill block melt spinning technique and the rapidly solidified microstructures were characterized using electron microscopy. The spinning technique and the rapidly solidified microstructures was assessed following heat treatments at 1033 and 1272 K. Rapidly solidified material of three alloys was reduced to particulate form and consolidated using hot isostatic pressing (HIP). The consolidated materials were also characterized using microscopy techniques. In order to evaluate the relative strengths of the consolidated alloys, compression tests were performed at room temperature and 1033 K on samples of as-HIPed and HIPed plus solution treated material. Yield strength, porosity, and oxidation resistance characteristics are given and compared.

Fabrication and optical characterization of imaging fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2005-09-15

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Imaging TiO2 nanoparticles on GaN nanowires with electrostatic force microscopy

NASA Astrophysics Data System (ADS)

Xie, Ting; Wen, Baomei; Liu, Guannan; Guo, Shiqi; Motayed, Abhishek; Murphy, Thomas; Gomez, R. D.

Gallium nitride (GaN) nanowires that are functionalized with metal-oxides nanoparticles have been explored extensively for gas sensing applications in the past few years. These sensors have several advantages over conventional schemes, including miniature size, low-power consumption and fast response and recovery times. The morphology of the oxide functionalization layer is critical to achieve faster response and recovery times, with the optimal size distribution of nanoparticles being in the range of 10 to 30 nm. However, it is challenging to characterize these nanoparticles on GaN nanowires using common techniques such as scanning electron microscopy, transmission electron microscopy, and x-ray diffraction. Here, we demonstrate electrostatic force microscopy in combination with atomic force microscopy as a non-destructive technique for morphological characterization of the dispersed TiO2 nanoparticles on GaN nanowires. We also discuss the applicability of this method to other material systems with a proposed tip-surface capacitor model. This project was sponsored through N5 Sensors and the Maryland Industrial Partnerships (MIPS, #5418).

Multiple exposure photographic (MEP) technique: an objective assessment of sperm motility in infertility management.

PubMed

Adetoro, O O

1988-06-01

Multiple exposure photography (MEP), an objective technique, was used in determining the percentage of motile sperms in the semen samples from 41 males being investigated for infertility. This technique was compared with the conventional subjective ordinary microscopy method of spermatozoal motility assessment. A satisfactory correlation was observed in percentage sperm motility assessment using the two methods but the MEP estimation was more consistent and reliable. The value of this technique of sperm motility study in the developing world is discussed.

Review of combined isotopic and optical nanoscopy

PubMed Central

Richter, Katharina N.; Rizzoli, Silvio O.; Jähne, Sebastian; Vogts, Angela; Lovric, Jelena

2017-01-01

Abstract. Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the “history” of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology. PMID:28466025

Co-axial Electrospun Polyacrylonitrile-Poly(methylmethacrylate) Nanofibers: Atomic Force Microscopy and Compositional Characterization

PubMed Central

Zander, N.E.; Strawhecker, K.E.; Orlicki, J.A.; Rawlett, A.M.; Beebe, T.P.

2011-01-01

Poly(methylmethacrylate) (PMMA)- Polyacrylonitrile (PAN) fibers were prepared using a conventional single-nozzle electrospinning technique. The as-spun fibers exhibited core-shell morphology as verified by transmission electron microscopy (TEM) and atomic force microscopy (AFM). AFM-phase and modulus mapping images of the fiber cross-section and x-ray photoelectron spectroscopy (XPS) analysis indicated PAN formed the shell and PMMA the core material. XPS, thermal gravimetric analysis (TGA), and elemental analysis were used to determine fiber compositional information. Soaking the fibers in solvent demonstrated removal of the core material, generating hollow PAN fibers. PMID:21928836

On-Chip Biomedical Imaging

PubMed Central

Göröcs, Zoltán; Ozcan, Aydogan

2012-01-01

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and micro electromechanical systems achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lensfree holographic microscopy, fluorescent on-chip microscopy and lensfree optical tomography. PMID:23558399

Evaluation of bleach-sedimentation for sterilising and concentrating Mycobacterium tuberculosis in sputum specimens

PubMed Central

2011-01-01

Background Bleach-sedimentation may improve microscopy for diagnosing tuberculosis by sterilising sputum and concentrating Mycobacterium tuberculosis. We studied gravity bleach-sedimentation effects on safety, sensitivity, speed and reliability of smear-microscopy. Methods This blinded, controlled study used sputum specimens (n = 72) from tuberculosis patients. Bleach concentrations and exposure times required to sterilise sputum (n = 31) were determined. In the light of these results, the performance of 5 gravity bleach-sedimentation techniques that sterilise sputum specimens (n = 16) were compared. The best-performing of these bleach-sedimentation techniques involved adding 1 volume of 5% bleach to 1 volume of sputum, shaking for 10-minutes, diluting in 8 volumes distilled water and sedimenting overnight before microscopy. This technique was further evaluated by comparing numbers of visible acid-fast bacilli, slide-reading speed and reliability for triplicate smears before versus after bleach-sedimentation of sputum specimens (n = 25). Triplicate smears were made to increase precision and were stained using the Ziehl-Neelsen method. Results M. tuberculosis in sputum was successfully sterilised by adding equal volumes of 15% bleach for 1-minute, 6% for 5-minutes or 3% for 20-minutes. Bleach-sedimentation significantly decreased the number of acid-fast bacilli visualised compared with conventional smears (geometric mean of acid-fast bacilli per 100 microscopy fields 166, 95%CI 68-406, versus 346, 95%CI 139-862, respectively; p = 0.02). Bleach-sedimentation diluted paucibacillary specimens less than specimens with higher concentrations of visible acid-fast bacilli (p = 0.02). Smears made from bleach-sedimented sputum were read more rapidly than conventional smears (9.6 versus 11.2 minutes, respectively, p = 0.03). Counting conventional acid-fast bacilli had high reliability (inter-observer agreement, r = 0.991) that was significantly reduced (p = 0.03) by bleach-sedimentation (to r = 0.707) because occasional strongly positive bleach-sedimented smears were misread as negative. Conclusions Gravity bleach-sedimentation improved laboratory safety by sterilising sputum but decreased the concentration of acid-fast bacilli visible on microscopy, especially for sputum specimens containing high concentrations of M. tuberculosis. Bleach-sedimentation allowed examination of more of each specimen in the time available but decreased the inter-observer reliability with which slides were read. Thus bleach-sedimentation effects vary depending upon specimen characteristics and whether microscopy was done for a specified time, or until a specified number of microscopy fields had been read. These findings provide an explanation for the contradictory results of previous studies. PMID:21985457

Evaluation of the interface between gutta-percha and two types of sealers using scanning electron microscopy (SEM).

PubMed

Eltair, Mohamed; Pitchika, Vinay; Hickel, Reinhard; Kühnisch, Jan; Diegritz, Christian

2018-05-01

The aim of the present study was to evaluate the adaptation of a calcium silicate bioceramic (BC) sealer with either BC or conventional gutta-percha compared with that of AH Plus sealer in different root canal sections. Seventy-two extracted mandibular premolars were divided randomly into six groups. After standardised chemomechanical preparation, four groups were obturated with the BC sealer and BC gutta-percha or conventional gutta-percha, and the other two groups were obturated with AH Plus sealer and conventional gutta-percha either in lateral compaction or in a single cone technique. Each root was sectioned into three sections. An impression was made from each section, and replicas were then made for scanning electron microscopy (SEM) analysis. Areas and interfacial gaps were identified using image analysis software. In addition to descriptive and explorative data analyses, linear regression analysis was performed. All specimens had measurable interfacial gaps. Significantly fewer gaps were found between conventional gutta-percha and sealer compared to those observed when using the BC gutta-percha (p < 0.001). However, minor interfacial gaps between sealer and dentin were observed with the BC sealer (p = 0.04). The technique of obturation in different root canal sections did not significantly affect the sealer adaptability. The type of gutta-percha as well as the sealer had a noticeable impact on the adaptability. Different obturation techniques will result in similar outcomes. However, within the limitations of the study, there seems to be no advantage in using the BC gutta-percha.

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

PubMed Central

Lavagnino, Zeno; Sancataldo, Giuseppe; d’Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca

2016-01-01

In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces. PMID:27033347

Digital stereo-holographic microscopy for studying three-dimensional particle dynamics

NASA Astrophysics Data System (ADS)

Byeon, Hyeokjun; Go, Taesik; Lee, Sang Joon

2018-06-01

A digital stereo-holographic microscopy (DsHM) with two viewing angles is proposed to measure 3D information of microscale particles. This approach includes two volumetric recordings and numerical reconstruction, and it involves the combination of separately reconstructed holograms. The 3D positional information of a particle was determined by searching the center of the overlapped reconstructed volume. After confirming the proposed technique using static spherical particles, the 3D information of moving particles suspended in a Hagen-Poiseiulle flow was successfully obtained. Moreover, the 3D information of nonspherical particles, including ellipsoidal particles and red blood cells, were measured using the proposed technique. In addition to 3D positional information, the orientation and shape of the test samples were obtained from the plane images by slicing the overlapped volume perpendicular to the directions of the image recordings. This DsHM technique will be useful in analyzing the 3D dynamic behavior of various nonspherical particles, which cannot be measured by conventional digital holographic microscopy.

Application of Optical Coherence Tomography Freeze-Drying Microscopy for Designing Lyophilization Process and Its Impact on Process Efficiency and Product Quality.

PubMed

Korang-Yeboah, Maxwell; Srinivasan, Charudharshini; Siddiqui, Akhtar; Awotwe-Otoo, David; Cruz, Celia N; Muhammad, Ashraf

2018-01-01

Optical coherence tomography freeze-drying microscopy (OCT-FDM) is a novel technique that allows the three-dimensional imaging of a drug product during the entire lyophilization process. OCT-FDM consists of a single-vial freeze dryer (SVFD) affixed with an optical coherence tomography (OCT) imaging system. Unlike the conventional techniques, such as modulated differential scanning calorimetry (mDSC) and light transmission freeze-drying microscopy, used for predicting the product collapse temperature (Tc), the OCT-FDM approach seeks to mimic the actual product and process conditions during the lyophilization process. However, there is limited understanding on the application of this emerging technique to the design of the lyophilization process. In this study, we investigated the suitability of OCT-FDM technique in designing a lyophilization process. Moreover, we compared the product quality attributes of the resulting lyophilized product manufactured using Tc, a critical process control parameter, as determined by OCT-FDM versus as estimated by mDSC. OCT-FDM analysis revealed the absence of collapse even for the low protein concentration (5 mg/ml) and low solid content formulation (1%w/v) studied. This was confirmed by lab scale lyophilization. In addition, lyophilization cycles designed using Tc values obtained from OCT-FDM were more efficient with higher sublimation rate and mass flux than the conventional cycles, since drying was conducted at higher shelf temperature. Finally, the quality attributes of the products lyophilized using Tc determined by OCT-FDM and mDSC were similar, and product shrinkage and cracks were observed in all the batches of freeze-dried products irrespective of the technique employed in predicting Tc.

Lens-free microscopy of cerebrospinal fluid for the laboratory diagnosis of meningitis

NASA Astrophysics Data System (ADS)

Delacroix, Robin; Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Blandin, Pierre; Dinten, Jean-Marc; Drancourt, Michel; Allier, Cédric

2018-02-01

The cytology of the cerebrospinal fluid is traditionally performed by an operator (physician, biologist) by means of a conventional light microscope. The operator visually counts the leukocytes (white blood cells) present in a sample of cerebrospinal fluid (10 μl). It is a tedious job and the result is operator-dependent. Here in order to circumvent the limitations of manual counting, we approach the question of numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis by means of lens-free microscopy. In a first step, a prospective counts of leukocytes was performed by five different operators using conventional optical microscopy. The visual counting yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm was adapted step-by-step for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes. The optimization of the automatic lens-free counting was based on the prospective analysis of 215 cerebrospinal fluid specimens. The optimized algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories.

High speed wavefront sensorless aberration correction in digital micromirror based confocal microscopy.

PubMed

Pozzi, P; Wilding, D; Soloviev, O; Verstraete, H; Bliek, L; Vdovin, G; Verhaegen, M

2017-01-23

The quality of fluorescence microscopy images is often impaired by the presence of sample induced optical aberrations. Adaptive optical elements such as deformable mirrors or spatial light modulators can be used to correct aberrations. However, previously reported techniques either require special sample preparation, or time consuming optimization procedures for the correction of static aberrations. This paper reports a technique for optical sectioning fluorescence microscopy capable of correcting dynamic aberrations in any fluorescent sample during the acquisition. This is achieved by implementing adaptive optics in a non conventional confocal microscopy setup, with multiple programmable confocal apertures, in which out of focus light can be separately detected, and used to optimize the correction performance with a sampling frequency an order of magnitude faster than the imaging rate of the system. The paper reports results comparing the correction performances to traditional image optimization algorithms, and demonstrates how the system can compensate for dynamic changes in the aberrations, such as those introduced during a focal stack acquisition though a thick sample.

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Depth-resolved cellular microrheology using HiLo microscopy.

PubMed

Michaelson, Jarett; Choi, Heejin; So, Peter; Huang, Hayden

2012-06-01

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties.

Cross-sectional TEM specimen preparation for W/B{sub 4}C multilayer sample using FIB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mondal, Puspen, E-mail: puspen@rrcat.gov.in; Pradhan, P. C.; Tiwari, Pragya

2016-05-23

A recent emergence of a cross-beam scanning electron microscopy (SEM)/focused-ion-beam (FIB) system have given choice to fabricate cross-sectional transmission electron microscopy (TEM) specimen of thin film multilayer sample. A 300 layer pair thin film multilayer sample of W/B{sub 4}C was used to demonstrate the specimen lift-out technique in very short time as compared to conventional cross-sectional sample preparation technique. To get large area electron transparent sample, sample prepared by FIB is followed by Ar{sup +} ion polishing at 2 kV with grazing incident. The prepared cross-sectional sample was characterized by transmission electron microscope.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Application of microscopy technique and high performance liquid chromatography for quality assessment of Polygonum multiflorum Thunb. (Heshouwu)

PubMed Central

Liang, Li; Zhao, Zhongzhen; Kang, Tingguo

2014-01-01

Background: The technique of microscopy has been applied for identification of Chinese materia medica (CMM) since decades. However, very few scientific publications report the combination of conventional microscopy and high performance liquid chromatography (HPLC) techniques for further application to quality assessment of CMM. Objective: The objective of this study is to analyze the quality of the dried root tuber of Polygonum multiflorum Thunb. (Heshouwu) and to establish the relationships between 2,3,5,4’-tetrahydroxystilbene-2-O-β-glucoside, combined anthraquinone (CAQ) and quantity of clusters of calcium oxalate. Materials and Methods: In this study, microscopy and HPLC techniques were applied to assess the quality of P. multiflorum Thunb., and SPSS software was used to establish the relationship between microscopic characteristics and chemical components. Results: The results showed close and direct correlations between the quantity of clusters of calcium oxalate in P. multiflorum Thunb. and the contents of 2,3,5,4’-tetrahydroxystilbene-2-O-β-glucoside and CAQ. From these results, it can be deduced that Polygoni Multiflori Radix with a higher quantity of clusters of calcium oxalate should be of better quality. Conclusion: The established method can be helpful for evaluating the quality of CMM based upon the identification and quantitation of chemical and ergastic substance of cells. PMID:25422540

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Whole-Brain Microscopy Meets In Vivo Neuroimaging: Techniques, Benefits, and Limitations.

PubMed

Aswendt, Markus; Schwarz, Martin; Abdelmoula, Walid M; Dijkstra, Jouke; Dedeurwaerdere, Stefanie

2017-02-01

Magnetic resonance imaging, positron emission tomography, and optical imaging have emerged as key tools to understand brain function and neurological disorders in preclinical mouse models. They offer the unique advantage of monitoring individual structural and functional changes over time. What remained unsolved until recently was to generate whole-brain microscopy data which can be correlated to the 3D in vivo neuroimaging data. Conventional histological sections are inappropriate especially for neuronal tracing or the unbiased screening for molecular targets through the whole brain. As part of the European Society for Molecular Imaging (ESMI) meeting 2016 in Utrecht, the Netherlands, we addressed this issue in the Molecular Neuroimaging study group meeting. Presentations covered new brain clearing methods, light sheet microscopes for large samples, and automatic registration of microscopy to in vivo imaging data. In this article, we summarize the discussion; give an overview of the novel techniques; and discuss the practical needs, benefits, and limitations.

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. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ocular Biocompatibility of Nitinol Intraocular Clips

PubMed Central

Velez-Montoya, Raul; Erlanger, Michael

2012-01-01

Purpose. To evaluate the tolerance and biocompatibility of a preformed nitinol intraocular clip in an animal model after anterior segment surgery. Methods. Yucatan mini-pigs were used. A 30-gauge prototype injector was used to attach a shape memory nitinol clip to the iris of five pigs. Another five eyes received conventional polypropylene suture with a modified Seipser slip knot. The authors compared the surgical time of each technique. All eyes underwent standard full-field electroretinogram at baseline and 8 weeks after surgery. The animals were euthanized and eyes collected for histologic analysis after 70 days (10 weeks) postsurgery. The corneal thickness, corneal endothelial cell counts, specular microscopy parameters, retina cell counts, and electroretinogram parameters were compared between the groups. A two sample t-test for means and a P value of 0.05 were use for assessing statistical differences between measurements. Results. The injection of the nitinol clip was 15 times faster than conventional suturing. There were no statistical differences between the groups for corneal thickness, endothelial cell counts, specular microscopy parameters, retina cell counts, and electroretinogram measurements. Conclusions. The nitinol clip prototype is well tolerated and showed no evidence of toxicity in the short-term. The injectable delivery system was faster and technically less challenging than conventional suture techniques. PMID:22064995

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

PubMed Central

Hanninen, Adam; Shu, Ming Wai; Potma, Eric O.

2017-01-01

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-μm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family. PMID:28966861

Increased numbers of Demodex in contact lens wearers.

PubMed

Jalbert, Isabelle; Rejab, Shazana

2015-06-01

The aim of this study was to determine if Demodex infestation is more frequent in contact lens wearers than in nonwearers. Secondary aims were to evaluate the effects of Demodex on the ocular surface (symptoms and signs) and to evaluate the ability of confocal laser scanning microscopy to detect and quantify the Demodex infestation compared with the conventional light microscopic technique. Forty Asian female participants (20 nonwearers, 20 lens wearers) with a mean (± SD) age of 27 (± 9) years were recruited. Ocular comfort scores (Ocular Surface Disease Index, Ocular Comfort Index, and Dry Eye Questionnaire), vital staining (corneal, conjunctival, and lid wiper), tear osmolarity, tear breakup time, and meibomian gland evaluation were evaluated. Demodex was detected using in vivo confocal microscopy and conventional light microscopy. The number of Demodex was higher in lens wearers than in nonwearers (7.6 [± 5.8] vs. 5.0 [± 3.1]; p = 0.02). Demodex was observed in a large majority (90%) of lens wearers and in 65% of nonwearers using confocal microscopy (p = 0.06). The detection rate was lower in both groups using conventional light microscopy (p = 0.003) where Demodex could only be confirmed in 70% and 60% of lens wearers and nonwearers, respectively. The number of Demodex tended to increase with age (ρ = 0.28, p = 0.08), but Demodex did not appear to affect ocular comfort or any clinical signs (p > 0.05). Contact lens wearers harbor Demodex as frequently as nonwearers and in higher numbers, which is best detected using in vivo confocal microscopy. The significance of these findings is uncertain because no associations were found with any symptoms and signs of dry eye disease.

Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

PubMed

von Olshausen, Philipp; Rohrbach, Alexander

2013-10-15

Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

Application of high-angle annular dark field scanning transmission electron microscopy, scanning transmission electron microscopy-energy dispersive X-ray spectrometry, and energy-filtered transmission electron microscopy to the characterization of nanoparticles in the environment.

PubMed

Utsunomiya, Satoshi; Ewing, Rodney C

2003-02-15

A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.

A phase space model of Fourier ptychographic microscopy

PubMed Central

Horstmeyer, Roarke; Yang, Changhuei

2014-01-01

A new computational imaging technique, termed Fourier ptychographic microscopy (FPM), uses a sequence of low-resolution images captured under varied illumination to iteratively converge upon a high-resolution complex sample estimate. Here, we propose a mathematical model of FPM that explicitly connects its operation to conventional ptychography, a common procedure applied to electron and X-ray diffractive imaging. Our mathematical framework demonstrates that under ideal illumination conditions, conventional ptychography and FPM both produce datasets that are mathematically linked by a linear transformation. We hope this finding encourages the future cross-pollination of ideas between two otherwise unconnected experimental imaging procedures. In addition, the coherence state of the illumination source used by each imaging platform is critical to successful operation, yet currently not well understood. We apply our mathematical framework to demonstrate that partial coherence uniquely alters both conventional ptychography’s and FPM’s captured data, but up to a certain threshold can still lead to accurate resolution-enhanced imaging through appropriate computational post-processing. We verify this theoretical finding through simulation and experiment. PMID:24514995

Programmable Colored Illumination Microscopy (PCIM): A practical and flexible optical staining approach for microscopic contrast enhancement

NASA Astrophysics Data System (ADS)

Zuo, Chao; Sun, Jiasong; Feng, Shijie; Hu, Yan; Chen, Qian

2016-03-01

Programmable colored illumination microscopy (PCIM) has been proposed as a flexible optical staining technique for microscopic contrast enhancement. In this method, we replace the condenser diaphragm of a conventional microscope with a programmable thin film transistor-liquid crystal display (TFT-LCD). By displaying different patterns on the LCD, numerous established imaging modalities can be realized, such as bright field, dark field, phase contrast, oblique illumination, and Rheinberg illuminations, which conventionally rely on intricate alterations in the respective microscope setups. Furthermore, the ease of modulating both the color and the intensity distribution at the aperture of the condenser opens the possibility to combine multiple microscopic techniques, or even realize completely new methods for optical color contrast staining, such as iridescent dark-field and iridescent phase-contrast imaging. The versatility and effectiveness of PCIM is demonstrated by imaging of several transparent colorless specimens, such as unstained lung cancer cells, diatom, textile fibers, and a cryosection of mouse kidney. Finally, the potentialities of PCIM for RGB-splitting imaging with stained samples are also explored by imaging stained red blood cells and a histological section.

Depth-resolved cellular microrheology using HiLo microscopy

PubMed Central

Michaelson, Jarett; Choi, Heejin; So, Peter; Huang, Hayden

2012-01-01

It is increasingly important to measure cell mechanical properties in three-dimensional environments. Particle tracking microrheology (PTM) can measure cellular viscoelastic properties; however, out-of-plane data can introduce artifacts into these measurements. We developed a technique that employs HiLo microscopy to reduce out-of-plane contributions. This method eliminated signals from 90% of probes 0.5 μm or further from the focal plane, while retaining all in-plane probes. We used this technique to characterize live-cell bilayers and found that there were significant, frequency-dependent changes to the extracted cell moduli when compared to conventional analysis. Our results indicate that removal of out-of-plane information is vital for accurate assessments of cell mechanical properties. PMID:22741071

Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies.

PubMed

Belsey, Natalie A; Garrett, Natalie L; Contreras-Rojas, L Rodrigo; Pickup-Gerlaugh, Adam J; Price, Gareth J; Moger, Julian; Guy, Richard H

2014-01-28

Stimulated Raman scattering microscopy was used to assess the permeation of topically applied drugs and formulation excipients into porcine skin. This chemically selective technique generates high-resolution 3D images, from which semi-quantitative information may be elucidated. Ibuprofen, applied as a close-to-saturated solution in propylene glycol, was directly observed to crystallise in/on the skin, as the co-solvent permeated more rapidly, resulting in precipitation of the drug. Coherent Raman scattering microscopy is also an excellent tool, in conjunction with more conventional confocal fluorescence microscopy, with which to image micro/nanoparticle-based formulations. Specifically, the uptake of particles into thermal ablation transport pathways in the skin has been examined. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-time high dynamic range laser scanning microscopy

NASA Astrophysics Data System (ADS)

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-04-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Structured illumination for wide-field Raman imaging of cell membranes

NASA Astrophysics Data System (ADS)

Chen, Houkai; Wang, Siqi; Zhang, Yuquan; Yang, Yong; Fang, Hui; Zhu, Siwei; Yuan, Xiaocong

2017-11-01

Although the diffraction limit still restricts their lateral resolution, conventional wide-field Raman imaging techniques offer fast imaging speeds compared with scanning schemes. To extend the lateral resolution of wide-field Raman microscopy using filters, standing-wave illumination technique is used, and an improvement of lateral resolution by a factor of more than two is achieved. Specifically, functionalized surface enhanced Raman scattering nanoparticles are employed to strengthen the desired scattering signals to label cell membranes. This wide-field Raman imaging technique affords various significant opportunities in the biological applications.

Transmitted light microscopy for visualizing the turbulent primary breakup of a microscale liquid jet

NASA Astrophysics Data System (ADS)

Reddemann, Manuel A.; Mathieu, Florian; Kneer, Reinhold

2013-11-01

Aiming at a maximum spatial resolution and a minimum motion blur, a new simple double-imaging transmitted light microscopy technique is developed in this work enabling a fundamental investigation of primary breakup of a microscale liquid jet. Contrary to conventional far-field visualization techniques, the working distance is minimized to increase the numerical aperture. The resulting images provide information about shapes, length scales and velocities of primary liquid structures. The method is applied to an optically dense spray leaving a 109-μm diesel nozzle at various injection pressures under atmospheric conditions. A phenomenological study on the temporal spray evolution is done with focus on droplet and ligament formation. Different breakup processes are identified and described. It is found that the jet is characterized by long ligaments parallel or angular to the inner jet region. These ligaments result from collapsing films developing at the spray edge. A significant influence of outlet velocity variation on shape and velocity of these ligaments is observed. The experimental results prove that a transmitted light microscopy technique with reduced working distance is an appropriate tool for a better understanding of primary breakup for small-scaled diesel nozzles and a valuable complement to highly complex measurement techniques.

The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

PubMed

Kirkbride, K Paul; Tridico, Silvana R

2010-02-25

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained. 2009 Elsevier Ireland Ltd. All rights reserved.

Force-detected nuclear magnetic resonance: recent advances and future challenges.

PubMed

Poggio, M; Degen, C L

2010-08-27

We review recent efforts to detect small numbers of nuclear spins using magnetic resonance force microscopy. Magnetic resonance force microscopy (MRFM) is a scanning probe technique that relies on the mechanical measurement of the weak magnetic force between a microscopic magnet and the magnetic moments in a sample. Spurred by the recent progress in fabricating ultrasensitive force detectors, MRFM has rapidly improved its capability over the last decade. Today it boasts a spin sensitivity that surpasses conventional, inductive nuclear magnetic resonance detectors by about eight orders of magnitude. In this review we touch on the origins of this technique and focus on its recent application to nanoscale nuclear spin ensembles, in particular on the imaging of nanoscale objects with a three-dimensional (3D) spatial resolution better than 10 nm. We consider the experimental advances driving this work and highlight the underlying physical principles and limitations of the method. Finally, we discuss the challenges that must be met in order to advance the technique towards single nuclear spin sensitivity-and perhaps-to 3D microscopy of molecules with atomic resolution.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Demosaiced pixel super-resolution in digital holography for multiplexed computational color imaging on-a-chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wu, Yichen; Zhang, Yibo; Luo, Wei; Ozcan, Aydogan

2017-03-01

Digital holographic on-chip microscopy achieves large space-bandwidth-products (e.g., >1 billion) by making use of pixel super-resolution techniques. To synthesize a digital holographic color image, one can take three sets of holograms representing the red (R), green (G) and blue (B) parts of the spectrum and digitally combine them to synthesize a color image. The data acquisition efficiency of this sequential illumination process can be improved by 3-fold using wavelength-multiplexed R, G and B illumination that simultaneously illuminates the sample, and using a Bayer color image sensor with known or calibrated transmission spectra to digitally demultiplex these three wavelength channels. This demultiplexing step is conventionally used with interpolation-based Bayer demosaicing methods. However, because the pixels of different color channels on a Bayer image sensor chip are not at the same physical location, conventional interpolation-based demosaicing process generates strong color artifacts, especially at rapidly oscillating hologram fringes, which become even more pronounced through digital wave propagation and phase retrieval processes. Here, we demonstrate that by merging the pixel super-resolution framework into the demultiplexing process, such color artifacts can be greatly suppressed. This novel technique, termed demosaiced pixel super-resolution (D-PSR) for digital holographic imaging, achieves very similar color imaging performance compared to conventional sequential R,G,B illumination, with 3-fold improvement in image acquisition time and data-efficiency. We successfully demonstrated the color imaging performance of this approach by imaging stained Pap smears. The D-PSR technique is broadly applicable to high-throughput, high-resolution digital holographic color microscopy techniques that can be used in resource-limited-settings and point-of-care offices.

Damage-free polymer surface modification employing inward-type plasma

NASA Astrophysics Data System (ADS)

Kanou, Ryo; Suga, Hiroshi; Utsumi, Hideyuki; Takahashi, Satoshi; Shirayama, Yuya; Watanabe, Norimichi; Petit, Stèphane; Shimizu, Tetsuo

2017-08-01

Inward-type plasmas, which spread upstream against the gas flow in the capillary tube where the gas is discharged, can react with samples placed near the entrance of such a capillary tube. In this study, surface modification of polymer surfaces is conducted using inward plasma. The modification is also done by conventional microplasma jet, and the modified surfaces with two plasma techniques are characterized by contact angle measurement, X-ray photoemission spectroscopy (XPS), and atomic force microscopy (AFM). Although inward-plasma-treated surfaces are less hydrophilic than conventional plasma-treated ones, they are still sufficiently hydrophilic for surface coatings. In addition, it turns out that the polymer surfaces irradiated with the inward plasma yield much smoother surfaces than those treated with the conventional plasma jet. Thus, the inward plasma treatment is a viable technique when the surface flatness is crucial, such as for the surface coating of plastic lenses.

Nanosilica coating for bonding improvements to zirconia.

PubMed

Chen, Chen; Chen, Gang; Xie, Haifeng; Dai, Wenyong; Zhang, Feimin

2013-01-01

Resin bonding to zirconia cannot be established from standard methods that are currently utilized in conventional silica-based dental ceramics. The solution-gelatin (sol-gel) process is a well developed silica-coating technique used to modify the surface of nonsilica-based ceramics. Here, we use this technique to improve resin bonding to zirconia, which we compared to zirconia surfaces treated with alumina sandblasting and tribochemical silica coating. We used the shear bond strength test to examine the effect of the various coatings on the short-term resin bonding of zirconia. Furthermore, we employed field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, and Fourier transform infrared spectroscopy to characterize the zirconia surfaces. Water-mist spraying was used to evaluate the durability of the coatings. To evaluate the biological safety of the experimental sol-gel silica coating, we conducted an in vitro Salmonella typhimurium reverse mutation assay (Ames mutagenicity test), cytotoxicity tests, and in vivo oral mucous membrane irritation tests. When compared to the conventional tribochemical silica coating, the experimental sol-gel silica coating provided the same shear bond strength, higher silicon contents, and better durability. Moreover, we observed no apparent mutagenicity, cytotoxicity, or irritation in this study. Therefore, the sol-gel technique represents a promising method for producing silica coatings on zirconia.

Nanosilica coating for bonding improvements to zirconia

PubMed Central

Chen, Chen; Chen, Gang; Xie, Haifeng; Dai, Wenyong; Zhang, Feimin

2013-01-01

Resin bonding to zirconia cannot be established from standard methods that are currently utilized in conventional silica-based dental ceramics. The solution–gelatin (sol–gel) process is a well developed silica-coating technique used to modify the surface of nonsilica-based ceramics. Here, we use this technique to improve resin bonding to zirconia, which we compared to zirconia surfaces treated with alumina sandblasting and tribochemical silica coating. We used the shear bond strength test to examine the effect of the various coatings on the short-term resin bonding of zirconia. Furthermore, we employed field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, atomic force microscopy, and Fourier transform infrared spectroscopy to characterize the zirconia surfaces. Water–mist spraying was used to evaluate the durability of the coatings. To evaluate the biological safety of the experimental sol–gel silica coating, we conducted an in vitro Salmonella typhimurium reverse mutation assay (Ames mutagenicity test), cytotoxicity tests, and in vivo oral mucous membrane irritation tests. When compared to the conventional tribochemical silica coating, the experimental sol–gel silica coating provided the same shear bond strength, higher silicon contents, and better durability. Moreover, we observed no apparent mutagenicity, cytotoxicity, or irritation in this study. Therefore, the sol–gel technique represents a promising method for producing silica coatings on zirconia. PMID:24179333

Towards real-time image deconvolution: application to confocal and STED microscopy

PubMed Central

Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

2013-01-01

Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

High-speed X-ray microscopy by use of high-resolution zone plates and synchrotron radiation.

PubMed

Hou, Qiyue; Wang, Zhili; Gao, Kun; Pan, Zhiyun; Wang, Dajiang; Ge, Xin; Zhang, Kai; Hong, Youli; Zhu, Peiping; Wu, Ziyu

2012-09-01

X-ray microscopy based on synchrotron radiation has become a fundamental tool in biology and life sciences to visualize the morphology of a specimen. These studies have particular requirements in terms of radiation damage and the image exposure time, which directly determines the total acquisition speed. To monitor and improve these key parameters, we present a novel X-ray microscopy method using a high-resolution zone plate as the objective and the matching condenser. Numerical simulations based on the scalar wave field theory validate the feasibility of the method and also indicate the performance of X-ray microscopy is optimized most with sub-10-nm-resolution zone plates. The proposed method is compatible with conventional X-ray microscopy techniques, such as computed tomography, and will find wide applications in time-resolved and/or dose-sensitive studies such as living cell imaging.

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

PubMed Central

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

2015-01-01

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

Friction force microscopy: a simple technique for identifying graphene on rough substrates and mapping the orientation of graphene grains on copper

NASA Astrophysics Data System (ADS)

Marsden, A. J.; Phillips, M.; Wilson, N. R.

2013-06-01

At a single atom thick, it is challenging to distinguish graphene from its substrate using conventional techniques. In this paper we show that friction force microscopy (FFM) is a simple and quick technique for identifying graphene on a range of samples, from growth substrates to rough insulators. We show that FFM is particularly effective for characterizing graphene grown on copper where it can correlate the graphene growth to the three-dimensional surface topography. Atomic lattice stick-slip friction is readily resolved and enables the crystallographic orientation of the graphene to be mapped nondestructively, reproducibly and at high resolution. We expect FFM to be similarly effective for studying graphene growth on other metal/locally crystalline substrates, including SiC, and for studying growth of other two-dimensional materials such as molybdenum disulfide and hexagonal boron nitride.

Preparation of herpes simplex virus-infected primary neurons for transmission electron microscopy.

PubMed

Miranda-Saksena, Monica; Boadle, Ross; Cunningham, Anthony L

2014-01-01

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction. Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus-cell interaction.

Measurement of Thermal Properties of Triticale Starch Films Using Photothermal Techniques

NASA Astrophysics Data System (ADS)

Correa-Pacheco, Z. N.; Cruz-Orea, A.; Jiménez-Pérez, J. L.; Solorzano-Ojeda, S. C.; Tramón-Pregnan, C. L.

2015-06-01

Nowadays, several commercially biodegradable materials have been developed with mechanical properties similar to those of conventional petrochemical-based polymers. These materials are made from renewable sources such as starch, cellulose, corn, and molasses, being very attractive for numerous applications in the plastics, food, and paper industries, among others. Starches from maize, rice, wheat, and potato are used in the food industry. However, other types of starches are not used due to their low protein content, such as triticale. In this study, starch films, processed using a single screw extruder with different compositions, were thermally and structurally characterized. The thermal diffusivity, thermal effusivity, and thermal conductivity of the biodegradable films were determined using photothermal techniques. The thermal diffusivity was measured using the open photoacoustic cell technique, and the thermal effusivity was obtained by the photopyroelectric technique in an inverse configuration. The results showed differences in thermal properties for the films. Also, the films microstructures were observed by scanning electron microscopy, transmission electron microscopy, and the crystalline structure determined by X-ray diffraction.

Biophotonic endoscopy: a review of clinical research techniques for optical imaging and sensing of early gastrointestinal cancer

PubMed Central

Coda, Sergio; Siersema, Peter D.; Stamp, Gordon W. H.; Thillainayagam, Andrew V.

2015-01-01

Detection, characterization, and staging constitute the fundamental elements in the endoscopic diagnosis of gastrointestinal diseases, but histology still remains the diagnostic gold standard. New developments in endoscopic techniques may challenge histopathology in the near future. An ideal endoscopic technique should combine a wide-field, “red flag” screening technique with an optical contrast or microscopy method for characterization and staging, all simultaneously available during the procedure. In theory, biophotonic advances have the potential to unite these elements to allow in vivo “optical biopsy.” These techniques may ultimately offer the potential to increase the rates of detection of high risk lesions and the ability to target biopsies and resections, and so reduce the need for biopsy, costs, and uncertainty for patients. However, their utility and sensitivity in clinical practice must be evaluated against those of conventional histopathology. This review describes some of the most recent applications of biophotonics in endoscopic optical imaging and metrology, along with their fundamental principles and the clinical experience that has been acquired in their deployment as tools for the endoscopist. Particular emphasis has been placed on translational label-free optical techniques, such as fluorescence spectroscopy, fluorescence lifetime imaging microscopy (FLIM), two-photon and multi-photon microscopy, second harmonic generation (SHG) and third harmonic generation (THG) imaging, optical coherence tomography (OCT), diffuse reflectance, Raman spectroscopy, and molecular imaging. PMID:26528489

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

PubMed

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

2011-10-01

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

Real-time high dynamic range laser scanning microscopy

PubMed Central

Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

2016-01-01

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging. PMID:27032979

Coherent Raman scattering microscopy for label-free imaging of live amphioxus

NASA Astrophysics Data System (ADS)

Yu, Zhilong; Chen, Tao; Zhang, Xiannian; Shen, Jie; Chen, Junyuan; Huang, Yanyi

2012-03-01

The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS) microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti- Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and native 3D optical sectioning ability.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

All-optical optoacoustic microscopy based on probe beam deflection technique.

PubMed

Maswadi, Saher M; Ibey, Bennett L; Roth, Caleb C; Tsyboulski, Dmitri A; Beier, Hope T; Glickman, Randolph D; Oraevsky, Alexander A

2016-09-01

Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 μV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

Characterization of new drug delivery nanosystems using atomic force microscopy

NASA Astrophysics Data System (ADS)

Spyratou, Ellas; Mourelatou, Elena A.; Demetzos, C.; Makropoulou, Mersini; Serafetinides, A. A.

2015-01-01

Liposomes are the most attractive lipid vesicles for targeted drug delivery in nanomedicine, behaving also as cell models in biophotonics research. The characterization of the micro-mechanical properties of drug carriers is an important issue and many analytical techniques are employed, as, for example, optical tweezers and atomic force microscopy. In this work, polyol hyperbranched polymers (HBPs) have been employed along with liposomes for the preparation of new chimeric advanced drug delivery nanosystems (Chi-aDDnSs). Aliphatic polyester HBPs with three different pseudogenerations G2, G3 and G4 with 16, 32, and 64 peripheral hydroxyl groups, respectively, have been incorporated in liposomal formulation. The atomic force microscopy (AFM) technique was used for the comparative study of the morphology and the mechanical properties of Chi-aDDnSs and conventional DDnS. The effects of both the HBPs architecture and the polyesters pseudogeneration number in the stability and the stiffness of chi-aDDnSs were examined. From the force-distance curves of AFM spectroscopy, the Young's modulus was calculated.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Birk, Udo J; Dobrucki, Jurek W; Cremer, Christoph

2016-05-01

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. Copyright © 2016. Published by Elsevier Inc.

Multielement mapping of alpha-SiC by scanning Auger microscopy

NASA Technical Reports Server (NTRS)

Browning, Ray; Smialek, James L.; Jacobson, Nathan S.

1987-01-01

Fine second-phase particles, numerous in sintered alpha-SiC, were analyzed by scanning Auger microscopy and conventional techniques. The Auger analysis utilized computer-controlled data acquisition, multielement correlation diagrams, and a high spatial resolution of 100 nm. This procedure enabled construction of false color maps and the detection of fine compositional details within these particles. Carbon, silicon oxide, and boron-rich particles (qualitatively as BN or B4C) predominated. The BN particles, sometimes having a carbon core, are believed to result from reaction between B4C additives and nitrogen sintering atmospheres.

Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging

NASA Technical Reports Server (NTRS)

Landis, W. J.; Hodgens, K. J.; Song, M. J.; Arena, J.; Kiyonaga, S.; Marko, M.; Owen, C.; McEwen, B. F.

1996-01-01

The interaction between collagen and mineral crystals in the normally calcifying leg tendons from the domestic turkey, Meleagris gallopavo, has been investigated at an ultrastructural level with conventional and high-voltage electron microscopy, computed tomography, and three-dimensional image reconstruction methods. Specimens treated by either aqueous or anhydrous techniques and resin-embedded were appropriately sectioned and regions of early tendon mineralization were photographed. On the basis of individual photomicrographs, stereoscopic pairs of images, and tomographic three-dimensional image reconstructions, platelet-shaped crystals may be demonstrated for the first time in association with the surface of collagen fibrils. Mineral is also observed in closely parallel arrays within collagen hole and overlap zones. The mineral deposition at these spatially distinct locations in the tendon provides insight into possible means by which calcification is mediated by collagen as a fundamental event in skeletal and dental formation among vertebrates.

STUDY ON SYNTHESIS AND EVOLUTION OF NANOCRYSTALLINE Mg4Ta2O9 BY AQUEOUS SOL-GEL PROCESS

NASA Astrophysics Data System (ADS)

Wu, H. T.; Yang, C. H.; Wu, W. B.; Yue, Y. L.

2012-06-01

Nanosized and highly reactive Mg4Ta2O9 were successfully synthesized by aqueous sol-gel method compared with conventional solid-state method. Ta-Mg-citric acid solution was first formed and then evaporated resulting in a dry gel for calcination in the temperature ranging from 600°C to 800°C for crystallization in oxygen atmosphere. The crystallization process from the gel to crystalline Mg4Ta2O9 was identified by thermal analysis and phase evolution of powders was studied using X-ray diffraction (XRD) technique during calcinations. Particle size and morphology were examined by transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HR-SEM). The results revealed that sol-gel process showed great advantages over conventional solid-state method and Mg4Ta2O9 nanopowders with the size of 20-30 nm were obtained at 800°C.

Three-dimensional microstructural characterization of bulk plutonium and uranium metals using focused ion beam technique

NASA Astrophysics Data System (ADS)

Chung, Brandon W.; Erler, Robert G.; Teslich, Nick E.

2016-05-01

Nuclear forensics requires accurate quantification of discriminating microstructural characteristics of the bulk nuclear material to identify its process history and provenance. Conventional metallographic preparation techniques for bulk plutonium (Pu) and uranium (U) metals are limited to providing information in two-dimension (2D) and do not allow for obtaining depth profile of the material. In this contribution, use of dual-beam focused ion-beam/scanning electron microscopy (FIB-SEM) to investigate the internal microstructure of bulk Pu and U metals is demonstrated. Our results demonstrate that the dual-beam methodology optimally elucidate microstructural features without preparation artifacts, and the three-dimensional (3D) characterization of inner microstructures can reveal salient microstructural features that cannot be observed from conventional metallographic techniques. Examples are shown to demonstrate the benefit of FIB-SEM in improving microstructural characterization of microscopic inclusions, particularly with respect to nuclear forensics.

Three-dimensional microstructural characterization of bulk plutonium and uranium metals using focused ion beam technique

DOE PAGES

Chung, Brandon W.; Erler, Robert G.; Teslich, Nick E.

2016-03-03

Nuclear forensics requires accurate quantification of discriminating microstructural characteristics of the bulk nuclear material to identify its process history and provenance. Conventional metallographic preparation techniques for bulk plutonium (Pu) and uranium (U) metals are limited to providing information in two-dimension (2D) and do not allow for obtaining depth profile of the material. In this contribution, use of dual-beam focused ion-beam/scanning electron microscopy (FIB-SEM) to investigate the internal microstructure of bulk Pu and U metals is demonstrated. Our results demonstrate that the dual-beam methodology optimally elucidate microstructural features without preparation artifacts, and the three-dimensional (3D) characterization of inner microstructures can revealmore » salient microstructural features that cannot be observed from conventional metallographic techniques. As a result, examples are shown to demonstrate the benefit of FIB-SEM in improving microstructural characterization of microscopic inclusions, particularly with respect to nuclear forensics.« less

Virtual microscopy: an evaluation of its validity and diagnostic performance in routine histologic diagnosis of skin tumors.

PubMed

Nielsen, Patricia Switten; Lindebjerg, Jan; Rasmussen, Jan; Starklint, Henrik; Waldstrøm, Marianne; Nielsen, Bjarne

2010-12-01

Digitization of histologic slides is associated with many advantages, and its use in routine diagnosis holds great promise. Nevertheless, few articles evaluate virtual microscopy in routine settings. This study is an evaluation of the validity and diagnostic performance of virtual microscopy in routine histologic diagnosis of skin tumors. Our aim is to investigate whether conventional microscopy of skin tumors can be replaced by virtual microscopy. Ninety-six skin tumors and skin-tumor-like changes were consecutively gathered over a 1-week period. Specimens were routinely processed, and digital slides were captured on Mirax Scan (Carl Zeiss MicroImaging, Göttingen, Germany). Four pathologists evaluated the 96 virtual slides and the associated 96 conventional slides twice with intermediate time intervals of at least 3 weeks. Virtual slides that caused difficulties were reevaluated to identify possible reasons for this. The accuracy was 89.2% for virtual microscopy and 92.7% for conventional microscopy. All κ coefficients expressed very good intra- and interobserver agreement. The sensitivities were 85.7% (78.0%-91.0%) and 92.0% (85.5%-95.7%) for virtual and conventional microscopy, respectively. The difference between the sensitivities was 6.3% (0.8%-12.6%). The subsequent reevaluation showed that virtual slides were as useful as conventional slides when rendering a diagnosis. Differences seen are presumed to be due to the pathologists' lack of experience using the virtual microscope. We conclude that it is feasible to make histologic diagnosis on the skin tumor types represented in this study using virtual microscopy after pathologists have completed a period of training. Larger studies should be conducted to verify whether virtual microscopy can replace conventional microscopy in routine practice. Copyright © 2010 Elsevier Inc. All rights reserved.

Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing

PubMed Central

Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G.; Menon, Rajesh; Gerton, Jordan M.; Jorgensen, Erik M.; Zalevsky, Zeev

2013-01-01

Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution. PMID:24466491

New method for characterizing paper coating structures using argon ion beam milling and field emission scanning electron microscopy.

PubMed

Dahlström, C; Allem, R; Uesaka, T

2011-02-01

We have developed a new method for characterizing microstructures of paper coating using argon ion beam milling technique and field emission scanning electron microscopy. The combination of these two techniques produces extremely high-quality images with very few artefacts, which are particularly suited for quantitative analyses of coating structures. A new evaluation method has been developed by using marker-controlled watershed segmentation technique of the secondary electron images. The high-quality secondary electron images with well-defined pores makes it possible to use this semi-automatic segmentation method. One advantage of using secondary electron images instead of backscattered electron images is being able to avoid possible overestimation of the porosity because of the signal depth. A comparison was made between the new method and the conventional method using greyscale histogram thresholding of backscattered electron images. The results showed that the conventional method overestimated the pore area by 20% and detected around 5% more pores than the new method. As examples of the application of the new method, we have investigated the distributions of coating binders, and the relationship between local coating porosity and base sheet structures. The technique revealed, for the first time with direct evidence, the long-suspected coating non-uniformity, i.e. binder migration, and the correlation between coating porosity versus base sheet mass density, in a straightforward way. © 2010 The Authors Journal compilation © 2010 The Royal Microscopical Society.

Nanoscale characterization of local structures and defects in photonic crystals using synchrotron-based transmission soft X-ray microscopy

PubMed Central

Nho, Hyun Woo; Kalegowda, Yogesh; Shin, Hyun-Joon; Yoon, Tae Hyun

2016-01-01

For the structural characterization of the polystyrene (PS)-based photonic crystals (PCs), fast and direct imaging capabilities of full field transmission X-ray microscopy (TXM) were demonstrated at soft X-ray energy. PS-based PCs were prepared on an O2-plasma treated Si3N4 window and their local structures and defects were investigated using this label-free TXM technique with an image acquisition speed of ~10 sec/frame and marginal radiation damage. Micro-domains of face-centered cubic (FCC (111)) and hexagonal close-packed (HCP (0001)) structures were dominantly found in PS-based PCs, while point and line defects, FCC (100), and 12-fold symmetry structures were also identified as minor components. Additionally, in situ observation capability for hydrated samples and 3D tomographic reconstruction of TXM images were also demonstrated. This soft X-ray full field TXM technique with faster image acquisition speed, in situ observation, and 3D tomography capability can be complementally used with the other X-ray microscopic techniques (i.e., scanning transmission X-ray microscopy, STXM) as well as conventional characterization methods (e.g., electron microscopic and optical/fluorescence microscopic techniques) for clearer structure identification of self-assembled PCs and better understanding of the relationship between their structures and resultant optical properties. PMID:27087141

Comparative catalytic reduction of 4-nitrophenol by polyacrylamide-gold nanocomposite synthesized by hydrothermal autoclaving and conventional heating routes

NASA Astrophysics Data System (ADS)

Salaheldin, Hosam I.

2017-12-01

Gold nanoparticles (AuNPs) in polymeric polyacrylamide (PAAm) matrix were synthesized using conventional heating and autoclaving thermal techniques. The synthesized Au/PAAm nanocomposite was characterized using UV-vis spectroscopy and high-resolution transmission electron microscopy. The size of the synthesized particles was approximately 6.37 nm and 8.19 nm with the conventional heating and autoclaving thermal techniques, respectively. Electron diffraction x-ray spectroscopy and the Fourier transformation infrared spectroscopy were used for the composition and elemental studies, which confirmed that the Au metallic atoms were synthesized and embedded within a PAAm matrix via a coordination bond between the carbonyl (-CONH2) group and the metallic NPs. X-ray diffraction confirmed the crystalline nature of the fabricated AuNPs with face centered cubic of nanocrystals. The catalytic activity of the as-prepared Au/PAAm nanocomposite for the reduction of 4-nitrophenol to 4-aminophenol was studied in the presence of sodium borohydrate. The synthesized AuNPs had an effective catalytic activity; the smaller NPs synthesized NPs with the conventional heating technique had a higher reaction kinetic rate in comparation with those synthesized with the autoclaving technique. Therefore, the Au/PAAm nanocomposite can be widely used as an eco-friendly, non-toxic, a fast and cost-effective product to remove versatile organic pollutants such as aromatic nitro compounds.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

USGS Publications Warehouse

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

Comparison of conventional and synchrotron-radiation-based microtomography of bone around dental implants

NASA Astrophysics Data System (ADS)

Cattaneo, Paolo M.; Dalstra, Michel; Beckmann, Felix; Donath, Tilman; Melsen, Birte

2004-10-01

This study explores the application of conventional micro tomography (μCT) and synchrotron radiation (SR) based μCT to evaluate the bone around titanium dental implants. The SR experiment was performed at beamline W2 of HASYLAB at DESY using a monochromatic X-ray beam of 50 keV. The testing material consisted of undecalcified bone segments harvested from the upper jaw of a macaca fascicularis monkey each containing a titanium dental implant. The results from the two different techniques were qualitatively compared with conventional histological sections examined under light microscopy. The SR-based μCT produced images that, especially at the bone-implant interface, are less noisy and sharper than the ones obtained with conventional μCT. For the proper evaluation of the implant-bone interface, only the SR-based μCT technique is able to display the areas of bony contact and visualize the true 3D structure of bone around dental implants correctly. This investigation shows that both conventional and SR-based μCT scanning techniques are non-destructive methods, which provide detailed images of bone. However with SR-based μCT it is possible to obtain an improved image quality of the bone surrounding dental implants, which display a level of detail comparable to histological sections. Therefore, SR-based μCT scanning could represent a valid, unbiased three-dimensional alternative to evaluate osseointegration of dental implants

Ex vivo confocal microscopy: a new diagnostic technique for mucormycosis.

PubMed

Leclercq, A; Cinotti, E; Labeille, B; Perrot, J L; Cambazard, F

2016-05-01

Skin-dedicated ex vivo confocal microscopy (EVCM) has so far mainly been employed to identify cutaneous tumours on freshly excised samples. We present two cases where EVCM has been used to diagnose cutaneous mucormycosis. The skin biopsies were evaluated by the skin-dedicated ex vivo confocal microscope VivaScope 2500(®) (MAVIG GmbH, Munich Germany) under both reflectance and fluorescence mode. Conventional direct optical examination on skin scraping and histological examination were later performed. Mucormycetes observed by EVCM presented as hyper-reflective elongated 20 μm in diameter structures with perpendicular ramifications. Fungi were found both under reflectance and fluorescence mode and were better visible with acridine orange under fluorescence EVCM. Conventional direct optical examination on skin scraping and histological examination found the same elongated and branching structures confirming the presence of Mucormycetes. Ex vivo confocal microscopy has both the advantages of being fast as the direct optical examination, and to be able to show the localisation of the fungi in the tissue like the histological examination. In our cases, EVCM allowed to rapidly confirm the clinical diagnosis of mucormycosis, which is essential for the treatment of this fungal infection. Further studies are needed to compare the performance of EVCM with the findings of conventional histological and mycological examinations. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Examining the contents of isolated Xenopus germinal vesicles.

PubMed

Gall, Joseph G; Wu, Zheng'an

2010-05-01

One can manually isolate the giant oocyte nucleus or germinal vesicle (GV) of Xenopus from a living oocyte with nothing more complicated than jewelers' forceps and a dissecting microscope. Similarly, one can remove the nuclear envelope by hand and allow the lampbrush chromosomes and other nuclear organelles to spread on a microscope slide. After centrifugation, the nuclear contents adhere tightly to the slide, where they can be subjected to immunostaining or fluorescent in situ hybridization for visualization by conventional or confocal microscopy. Preparations of isolated GV contents reveal details of nuclear structure that are almost impossible to attain by more conventional techniques.

Expansion Mini-Microscopy: An Enabling Alternative in Point-of-Care Diagnostics

PubMed Central

Zhang, Yu Shrike; Santiago, Grissel Trujillo-de; Alvarez, Mario Moisés; Schiff, Steven J.; Boyden, Edward S.; Khademhosseini, Ali

2017-01-01

Diagnostics play a significant role in health care. In the developing world and low-resource regions the utility for point-of-care (POC) diagnostics becomes even greater. This need has long been recognized, and diagnostic technology has seen tremendous progress with the development of portable instrumentation such as miniature imagers featuring low complexity and cost. However, such inexpensive devices have not been able to achieve a resolution sufficient for POC detection of pathogens at very small scales, such as single-cell parasites, bacteria, fungi, and viruses. To this end, expansion microscopy (ExM) is a recently developed technique that, by physically expanding preserved biological specimens through a chemical process, enables super-resolution imaging on conventional microscopes and improves imaging resolution of a given microscope without the need to modify the existing microscope hardware. Here we review recent advances in ExM and portable imagers, respectively, and discuss the rational combination of the two technologies, that we term expansion mini-microscopy (ExMM). In ExMM, the physical expansion of a biological sample followed by imaging on a mini-microscope achieves a resolution as high as that attainable by conventional high-end microscopes imaging non-expanded samples, at significant reduction in cost. We believe that this newly developed ExMM technique is likely to find widespread applications in POC diagnostics in resource-limited and remote regions by expanded-scale imaging of biological specimens that are otherwise not resolvable using low-cost imagers. PMID:29062977

Imaging of Cell-Cell Communication in a Vertical Orientation Reveals High-Resolution Structure of Immunological Synapse and Novel PD-1 Dynamics

PubMed Central

Jang, Joon Hee; Huang, Yu; Zheng, Peilin; Jo, Myeong Chan; Bertolet, Grant; Qin, Lidong; Liu, Dongfang

2015-01-01

The immunological synapse (IS) is one of the most pivotal communication strategies in immune cells. Understanding the molecular basis of the IS provides critical information regarding how immune cells mount an effective immune response. Fluorescence microscopy provides a fundamental tool to study the IS. However, current imaging techniques for studying the IS cannot sufficiently achieve high resolution in real cell-cell conjugates. Here we present a new device that allows for high-resolution imaging of the IS with conventional confocal microscopy in a high-throughput manner. Combining micropits and single cell trap arrays, we have developed a new microfluidic platform that allows visualization of the IS in vertically “stacked” cells. Using this vertical cell pairing (VCP) system, we investigated the dynamics of the inhibitory synapse mediated by an inhibitory receptor, programed death protein-1 (PD-1) and the cytotoxic synapse at the single cell level. In addition to the technique innovation, we demonstrated novel biological findings by this VCP device, including novel distribution of F-actin and cytolytic granules at the IS, PD-1 microclusters in the NK IS, and kinetics of cytotoxicity. We propose that this high-throughput, cost-effective, easy-to-use VCP system, along with conventional imaging techniques, can be used to address a number of significant biological questions in a variety of disciplines. PMID:26123352

Electrochemical characterization of pulsed layer deposited hydroxyapatite-zirconia layers on Ti-21Nb-15Ta-6Zr alloy for biomedical application

NASA Astrophysics Data System (ADS)

Izquierdo, Javier; Bolat, Georgiana; Cimpoesu, Nicanor; Trinca, Lucia Carmen; Mareci, Daniel; Souto, Ricardo Manuel

2016-11-01

A new titanium base Ti-21Nb-15Ta-6Zr alloy covered with hydroxyapatite-zirconia (HA-ZrO2) by pulsed laser deposition (PLD) technique was characterized regarding its corrosion resistance in simulated physiological Ringer's solution at 37 °C. For the sake of comparison, Ti-6Al-4V standard implant alloy, with and without hydroxyapatite-zirconia coating, was also characterized. Multiscale electrochemical analysis using both conventional averaging electrochemical techniques, namely electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization, and spatially-resolved microelectrochemical techniques (scanning electrochemical microscopy, SECM) were used to investigate the electrochemical behaviour of the materials. In addition, scanning electron microscopy evidenced that no relevant surface morphology changes occurred on the materials upon immersion in the simulated physiological solution, despite variations in their electrochemical behaviour. Although uncoated metals appear to show better performances during conventional corrosion tests, the response is still quite similar for the HA-ZrO2 coated materials while providing superior resistance towards electron transfer due to the formation of a more dense film on the surface, thus effectively behaving as a passive material. It is believed corrosion of the HA-ZrO2 coated Ti-21Nb-15Ta-6Zr alloy will have negligible effect upon biochemical and cellular events at the bone-implant interface and could facilitate osseointegration.

Multiple speckle illumination for optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Poisson, Florian; Stasio, Nicolino; Moser, Christophe; Psaltis, Demetri; Bossy, Emmanuel

2017-03-01

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,1, 2 and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:3 in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.2

Super-Resolution Microscopy Techniques and Their Potential for Applications in Radiation Biophysics.

PubMed

Eberle, Jan Philipp; Rapp, Alexander; Krufczik, Matthias; Eryilmaz, Marion; Gunkel, Manuel; Erfle, Holger; Hausmann, Michael

2017-01-01

Fluorescence microscopy is an essential tool for imaging tagged biological structures. Due to the wave nature of light, the resolution of a conventional fluorescence microscope is limited laterally to about 200 nm and axially to about 600 nm, which is often referred to as the Abbe limit. This hampers the observation of important biological structures and dynamics in the nano-scaled range ~10 nm to ~100 nm. Consequentially, various methods have been developed circumventing this limit of resolution. Super-resolution microscopy comprises several of those methods employing physical and/or chemical properties, such as optical/instrumental modifications and specific labeling of samples. In this article, we will give a brief insight into a variety of selected optical microscopy methods reaching super-resolution beyond the Abbe limit. We will survey three different concepts in connection to biological applications in radiation research without making a claim to be complete.

Electron microscopy approach for the visualization of the epithelial and endothelial glycocalyx.

PubMed

Chevalier, L; Selim, J; Genty, D; Baste, J M; Piton, N; Boukhalfa, I; Hamzaoui, M; Pareige, P; Richard, V

2017-06-01

This study presents a methodological approach for the visualization of the glycocalyx by electron microscopy. The glycocalyx is a three dimensional network mainly composed of glycolipids, glycoproteins and proteoglycans associated with the plasma membrane. Since less than a decade, the epithelial and endothelial glycocalyx proved to play an important role in physiology and pathology, increasing its research interest especially in vascular functions. Therefore, visualization of the glycocalyx requires reliable techniques and its preservation remains challenging due to its fragile and dynamic organization, which is highly sensitive to the different process steps for electron microscopy sampling. In this study, chemical fixation was performed by perfusion as a good alternative to conventional fixation. Additional lanthanum nitrate in the fixative enhances staining of the glycocalyx in transmission electron microscopy bright field and improves its visualization by detecting the elastic scattered electrons, thus providing a chemical contrast. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

PubMed Central

Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

2012-01-01

Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

PubMed

Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

2012-11-01

Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urs, Necdet Onur; Mozooni, Babak; Kustov, Mikhail

2016-05-15

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.more » Beyond conventional domain imaging, the use of a magneto-optical indicator technique for local temperature sensing is shown.« less

Magnetic tweezers for the measurement of twist and torque.

PubMed

Lipfert, Jan; Lee, Mina; Ordu, Orkide; Kerssemakers, Jacob W J; Dekker, Nynke H

2014-05-19

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a "conventional" magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the "conventional" magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Nanoparticles as smart treatment-delivery systems in plants: assessment of different techniques of microscopy for their visualization in plant tissues.

PubMed

González-Melendi, P; Fernández-Pacheco, R; Coronado, M J; Corredor, E; Testillano, P S; Risueño, M C; Marquina, C; Ibarra, M R; Rubiales, D; Pérez-de-Luque, A

2008-01-01

The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies.

Nanoparticles as Smart Treatment-delivery Systems in Plants: Assessment of Different Techniques of Microscopy for their Visualization in Plant Tissues

PubMed Central

González-Melendi, P.; Fernández-Pacheco, R.; Coronado, M. J.; Corredor, E.; Testillano, P. S.; Risueño, M. C.; Marquina, C.; Ibarra, M. R.; Rubiales, D.; Pérez-de-Luque, A.

2008-01-01

Background and Aims The great potential of using nanodevices as delivery systems to specific targets in living organisms was first explored for medical uses. In plants, the same principles can be applied for a broad range of uses, in particular to tackle infections. Nanoparticles tagged to agrochemicals or other substances could reduce the damage to other plant tissues and the amount of chemicals released into the environment. To explore the benefits of applying nanotechnology to agriculture, the first stage is to work out the correct penetration and transport of the nanoparticles into plants. This research is aimed (a) to put forward a number of tools for the detection and analysis of core-shell magnetic nanoparticles introduced into plants and (b) to assess the use of such magnetic nanoparticles for their concentration in selected plant tissues by magnetic field gradients. Methods Cucurbita pepo plants were cultivated in vitro and treated with carbon-coated Fe nanoparticles. Different microscopy techniques were used for the detection and analysis of these magnetic nanoparticles, ranging from conventional light microscopy to confocal and electron microscopy. Key Results Penetration and translocation of magnetic nanoparticles in whole living plants and into plant cells were determined. The magnetic character allowed nanoparticles to be positioned in the desired plant tissue by applying a magnetic field gradient there; also the graphitic shell made good visualization possible using different microscopy techniques. Conclusions The results open a wide range of possibilities for using magnetic nanoparticles in general plant research and agronomy. The nanoparticles can be charged with different substances, introduced within the plants and, if necessary, concentrated into localized areas by using magnets. Also simple or more complex microscopical techniques can be used in localization studies. PMID:17998213

Autofocusing Airy beam STED microscopy with long focal distance

NASA Astrophysics Data System (ADS)

Hu, Di; Liang, Yao; Chen, Yin; Chen, Zan Hui; Huang, Xu Guang

2017-12-01

Stimulated emission depletion (STED) is a very important technique in super-resolution microscopy. Until now, while autofocusing Airy beam (AAB) has been an attractive theme for both theoretical and applied researches, there are almost no report on AABs being used in STED microscopy. In this paper, we propose a novel STED microscopy based on AABs. A radially symmetric 3/2 phase plate is involved to simultaneously generate autofocusing excitation- and depletion-Airy beams. Remarkably, the AAB can auto-focus to a wavelength-scale spot with a long focal depth (several millimeters): on the contrary, the working distance of a conventional high numerical aperture (NA) objective is usually very short (about 200 μm). Our calculations indicate that the AAB based STED microscopy can achieve a super-resolution spot with FWHM of 58 nm while the focal length is 4.638 mm. Moreover, with properties of non-diffracting and self-healing, the Airy beam could enable a reduction of the scattering distortion induced by the specimens and has a great potential in imaging thick specimens.

Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining.

PubMed Central

Sherburne, R; Armstrong, G D

1989-01-01

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques. Images PMID:2570062

Correction of stain variations in nuclear refractive index of clinical histology specimens

PubMed Central

Uttam, Shikhar; Bista, Rajan K.; Hartman, Douglas J.; Brand, Randall E.; Liu, Yang

2011-01-01

For any technique to be adopted into a clinical setting, it is imperative that it seamlessly integrates with well-established clinical diagnostic workflow. We recently developed an optical microscopy technique—spatial-domain low-coherence quantitative phase microscopy (SL-QPM) that can extract the refractive index of the cell nucleus from the standard histology specimens on glass slides prepared via standard clinical protocols. This technique has shown great potential in detecting cancer with a better sensitivity than conventional pathology. A major hurdle in the clinical translation of this technique is the intrinsic variation among staining agents used in histology specimens, which limits the accuracy of refractive index measurements of clinical samples. In this paper, we present a simple and easily generalizable method to remove the effect of variations in staining levels on nuclear refractive index obtained with SL-QPM. We illustrate the efficacy of our correction method by applying it to variously stained histology samples from animal model and clinical specimens. PMID:22112118

High-speed transport-of-intensity phase microscopy with an electrically tunable lens.

PubMed

Zuo, Chao; Chen, Qian; Qu, Weijuan; Asundi, Anand

2013-10-07

We present a high-speed transport-of-intensity equation (TIE) quantitative phase microscopy technique, named TL-TIE, by combining an electrically tunable lens with a conventional transmission microscope. This permits the specimen at different focus position to be imaged in rapid succession, with constant magnification and no physically moving parts. The simplified image stack collection significantly reduces the acquisition time, allows for the diffraction-limited through-focus intensity stack collection at 15 frames per second, making dynamic TIE phase imaging possible. The technique is demonstrated by profiling of microlens array using optimal frequency selection scheme, and time-lapse imaging of live breast cancer cells by inversion the defocused phase optical transfer function to correct the phase blurring in traditional TIE. Experimental results illustrate its outstanding capability of the technique for quantitative phase imaging, through a simple, non-interferometric, high-speed, high-resolution, and unwrapping-free approach with prosperous applications in micro-optics, life sciences and bio-photonics.

Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections.

PubMed

Meza, Daphne; Wang, Danni; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-04-01

Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy. Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality). HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast. Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning. © 2015 Wiley Periodicals, Inc.

Digital microscopy as valid alternative to conventional microscopy for histological evaluation of Barrett's esophagus biopsies.

PubMed

van der Wel, M J; Duits, L C; Seldenrijk, C A; Offerhaus, G J; Visser, M; Ten Kate, F J; de Boer, O J; Tijssen, J G; Bergman, J J; Meijer, S L

2017-11-01

Management of Barrett's esophagus (BE) relies heavily on histopathological assessment of biopsies, associated with significant intra- and interobserver variability. Guidelines recommend biopsy review by an expert in case of dysplasia. Conventional review of biopsies, however, is impractical and does not allow for teleconferencing or annotations. An expert digital review platform might overcome these limitations. We compared diagnostic agreement of digital and conventional microscopy for diagnosing BE ± dysplasia. Sixty BE biopsy glass slides (non-dysplastic BE (NDBE); n = 25, low-grade dysplasia (LGD); n = 20; high-grade dysplasia (HGD); n = 15) were scanned at ×20 magnification. The slides were assessed four times by five expert BE pathologists, all practicing histopathologists (range: 5-30 years), in 2 alternating rounds of digital and conventional microscopy, each in randomized order and sequence of slides. Intraobserver and pairwise interobserver agreement were calculated, using custom weighted Cohen's kappa, adjusted for the maximum possible kappa scores. Split into three categories (NDBE, IND, LGD+HGD), the mean intraobserver agreement was 0.75 and 0.84 for digital and conventional assessment, respectively (p = 0.35). Mean pairwise interobserver agreement was 0.80 for digital and 0.85 for conventional microscopy (p = 0.17). In 47/60 (78%) of digital microscopy reviews a majority vote of ≥3 pathologists was reached before consensus meeting. After group discussion, a majority vote was achieved in all cases (60/60). Diagnostic agreement of digital microscopy is comparable to that of conventional microscopy. These outcomes justify the use of digital slides in a nationwide, web-based BE revision platform in the Netherlands. This will overcome the practical issues associated with conventional histologic review by multiple pathologists. © The Authors 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Magnetic Resonance Microscopy of the Lung

NASA Astrophysics Data System (ADS)

Johnson, G. Allan

1999-11-01

The lung presents both challenges and opportunities for study by magnetic resonance imaging (MRI). The technical challenges arise from respiratory and cardiac motion, limited signal from the tissues, and unique physical structure of the lung. These challenges are heightened in magnetic resonance microscopy (MRM) where the spatial resolution may be up to a million times higher than that of conventional MRI. The development of successful techniques for MRM of the lung present enormous opportunities for basic studies of lung structure and function, toxicology, environmental stress, and drug discovery by permitting investigators to study this most essential organ nondestructively in the live animal. Over the last 15 years, scientists at the Duke Center for In Vivo Microscopy have developed techniques for MRM in the live animal through an interdisciplinary program of biology, physics, chemistry, electrical engineering, and computer science. This talk will focus on the development of specialized radiofrequency coils for lung imaging, projection encoding methods to limit susceptibility losses, specialized support structures to control and monitor physiologic motion, and the most recent development of hyperpolarized gas imaging with ^3He and ^129Xe.

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

PubMed Central

Glaser, Adam K.; Chen, Ye; Liu, Jonathan T.C.

2017-01-01

Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method’s flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy. PMID:28983499

Diagnostic efficacy of microscopy, rapid diagnostic test and polymerase chain reaction for malaria using bayesian latent class analysis.

PubMed

Saha, Sreemanti; Narang, Rahul; Deshmukh, Pradeep; Pote, Kiran; Anvikar, Anup; Narang, Pratibha

2017-01-01

The diagnostic techniques for malaria are undergoing a change depending on the availability of newer diagnostics and annual parasite index of infection in a particular area. At the country level, guidelines are available for selection of diagnostic tests; however, at the local level, this decision is made based on malaria situation in the area. The tests are evaluated against the gold standard, and if that standard has limitations, it becomes difficult to compare other available tests. Bayesian latent class analysis computes its internal standard rather than using the conventional gold standard and helps comparison of various tests including the conventional gold standard. In a cross-sectional study conducted in a tertiary care hospital setting, we have evaluated smear microscopy, rapid diagnostic test (RDT), and polymerase chain reaction (PCR) for diagnosis of malaria using Bayesian latent class analysis. We found the magnitude of malaria to be 17.7% (95% confidence interval: 12.5%-23.9%) among the study subjects. In the present study, the sensitivity of microscopy was 63%, but it had very high specificity (99.4%). Sensitivity and specificity of RDT and PCR were high with RDT having a marginally higher sensitivity (94% vs. 90%) and specificity (99% vs. 95%). On comparison of likelihood ratios (LRs), RDT had the highest LR for positive test result (175) and the lowest LR for negative test result (0.058) among the three tests. In settings like ours conventional smear microscopy may be replaced with RDT and as we move toward elimination and facilities become available PCR may be roped into detect cases with lower parasitaemia.

A simple backscattering microscope for fast tracking of biological molecules

PubMed Central

Sowa, Yoshiyuki; Steel, Bradley C.; Berry, Richard M.

2010-01-01

Recent developments in techniques for observing single molecules under light microscopes have helped reveal the mechanisms by which molecular machines work. A wide range of markers can be used to detect molecules, from single fluorophores to micron sized markers, depending on the research interest. Here, we present a new and simple objective-type backscattering microscope to track gold nanoparticles with nanometer and microsecond resolution. The total noise of our system in a 55 kHz bandwidth is ∼0.6 nm per axis, sufficient to measure molecular movement. We found our backscattering microscopy to be useful not only for in vitro but also for in vivo experiments because of lower background scattering from cells than in conventional dark-field microscopy. We demonstrate the application of this technique to measuring the motion of a biological rotary molecular motor, the bacterial flagellar motor, in live Escherichia coli cells. PMID:21133475

Topography and Mechanical Property Mapping of International Simple Glass Surfaces with Atomic Force Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, Eric M

2014-01-01

Quantitative Nanomechanical Peak Force (PF-QNM) TappingModeTM atomic force microscopy measurements are presented for the first time on polished glass surfaces. The PF-QNM technique allows for topography and mechanical property information to be measured simultaneously at each pixel. Results for the international simple glass which represents a simplified version of SON68 glass suggests an average Young s modulus of 78.8 15.1 GPa is within the experimental error of the modulus measured for SON68 glass (83.6 2 GPa) with conventional approaches. Application of the PF-QNM technique will be extended to in situ glass corrosion experiments with the goal of gaining atomic-scale insightsmore » into altered layer development by exploiting the mechanical property differences that exist between silica gel (e.g., altered layer) and pristine glass surface.« less

Dimensional metrology of lab-on-a-chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy.

PubMed

Reyes, D R; Halter, M; Hwang, J

2015-07-01

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in-house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser-scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three-dimensional metrology of critical internal structures in lab-on-a-chip devices because scans can be performed rapidly and noninvasively prior to their use. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, Thorben

2015-11-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

ATOMIC RESOLUTION CRYO ELECTRON MICROSCOPY OF MACROMOLECULAR COMPLEXES

PubMed Central

ZHOU, Z. HONG

2013-01-01

Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines. PMID:21501817

Super-resolution fluorescence microscopy by stepwise optical saturation

PubMed Central

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

Contact resonance atomic force microscopy imaging in air and water using photothermal excitation.

PubMed

Kocun, Marta; Labuda, Aleksander; Gannepalli, Anil; Proksch, Roger

2015-08-01

Contact Resonance Force Microscopy (CR-FM) is a leading atomic force microscopy technique for measuring viscoelastic nano-mechanical properties. Conventional piezo-excited CR-FM measurements have been limited to imaging in air, since the "forest of peaks" frequency response associated with acoustic excitation methods effectively masks the true cantilever resonance. Using photothermal excitation results in clean contact, resonance spectra that closely match the ideal frequency response of the cantilever, allowing unambiguous and simple resonance frequency and quality factor measurements in air and liquids alike. This extends the capabilities of CR-FM to biologically relevant and other soft samples in liquid environments. We demonstrate CR-FM in air and water on both stiff silicon/titanium samples and softer polystyrene-polyethylene-polypropylene polymer samples with the quantitative moduli having very good agreement between expected and measured values.

Single- and multi-frequency detection of surface displacements via scanning probe microscopy.

PubMed

Romanyuk, Konstantin; Luchkin, Sergey Yu; Ivanov, Maxim; Kalinin, Arseny; Kholkin, Andrei L

2015-02-01

Piezoresponse force microscopy (PFM) provides a novel opportunity to detect picometer-level displacements induced by an electric field applied through a conducting tip of an atomic force microscope (AFM). Recently, it was discovered that superb vertical sensitivity provided by PFM is high enough to monitor electric-field-induced ionic displacements in solids, the technique being referred to as electrochemical strain microscopy (ESM). ESM has been implemented only in multi-frequency detection modes such as dual AC resonance tracking (DART) and band excitation, where the response is recorded within a finite frequency range, typically around the first contact resonance. In this paper, we analyze and compare signal-to-noise ratios of the conventional single-frequency method with multi-frequency regimes of measuring surface displacements. Single-frequency detection ESM is demonstrated using a commercial AFM.

[Surface characteristics of the acrylic resins according to the polishing methods].

PubMed

Vitalariu, Anca Mihaela; Lazăr, Lenuţa; Buruiană, Tinca; Diaconu, Diana; Tatarciuc, Monica Silvia

2011-01-01

The objective of this study was to evaluate the effect of the polishing technique and glazing on the porosity of the dental resins. The studied resins were: Castapress/Vertex, Prothyl Hot/Zermack, Rapid Simplified/Vertex, Duracryl Plus/Spofa Dental, Vertex-Soft/Vertex, Superacryl Plus/Spofa Dental. Thirty specimens, five for every resin, of 50/25/2 cm in size were done. One surface of each sample was polished with extrahard tungsten carbide burs, the other surface being polished with extrahard extrafine and diamond burs. The final polishing was done using a conventional method: pumice, water and lathe bristle brush for 90 seconds, 1500 rpm and soft leather polishing wheel for 90 seconds, 3000 rpm. Twenty surfaces were glazed after polishing with Glaze/Bosworth. Vertex Soft specimens were not polished because this is a resilient material. Surface porosity of the acrylic resin specimens was measured by optical microscopy. The lowest porosity was obtained by conventional polishing combined with glazing techniques. No differences between glazed and non/glazed self-curing resin specimens were noticed, but there were differences between self-curing and heat-curing resins. Conventional lathe polishing method is an effective and reliable technique for polishing dental resins. Specimens of self-curing resin had a higher porosity than heat curing resin following the same surface treatment. Higher surface smoothness was obtained by conventional lathe polishing completed by glazing.

Virtual Microscopy in Histopathology Training: Changing Student Attitudes in 3 Successive Academic Years.

PubMed

Bertram, Christof A; Firsching, Theresa; Klopfleisch, Robert

2018-01-01

Several veterinary faculties have integrated virtual microscopy into their curricula in recent years to improve and refine their teaching techniques. The many advantages of this recent technology are described in the literature, including remote access and an equal and constant slide quality for all students. However, no study has analyzed the change of perception toward virtual microscopy at different time points of students' academic educations. In the present study, veterinary students in 3 academic years were asked for their perspectives and attitudes toward virtual microscopy and conventional light microscopy. Third-, fourth-, and fifth-year veterinary students filled out a questionnaire with 12 questions. The answers revealed that virtual microscopy was overall well accepted by students of all academic years. Most students even suggested that virtual microscopy be implemented more extensively as the modality for final histopathology examinations. Nevertheless, training in the use of light microscopy and associated skills was surprisingly well appreciated. Regardless of their academic year, most students considered these skills important and necessary, and they felt that light microscopy should not be completely replaced. The reasons for this view differed depending on academic year, as the perceived main disadvantage of virtual microscopy varied. Third-year students feared that they would not acquire sufficient light microscopy skills. Fifth-year students considered technical difficulties (i.e., insufficient transmission speed) to be the main disadvantage of this newer teaching modality.

Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules.

PubMed

Burnette, Dylan T; Sengupta, Prabuddha; Dai, Yuhai; Lippincott-Schwartz, Jennifer; Kachar, Bechara

2011-12-27

Superresolution imaging techniques based on the precise localization of single molecules, such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), achieve high resolution by fitting images of single fluorescent molecules with a theoretical Gaussian to localize them with a precision on the order of tens of nanometers. PALM/STORM rely on photoactivated proteins or photoswitching dyes, respectively, which makes them technically challenging. We present a simple and practical way of producing point localization-based superresolution images that does not require photoactivatable or photoswitching probes. Called bleaching/blinking assisted localization microscopy (BaLM), the technique relies on the intrinsic bleaching and blinking behaviors characteristic of all commonly used fluorescent probes. To detect single fluorophores, we simply acquire a stream of fluorescence images. Fluorophore bleach or blink-off events are detected by subtracting from each image of the series the subsequent image. Similarly, blink-on events are detected by subtracting from each frame the previous one. After image subtractions, fluorescence emission signals from single fluorophores are identified and the localizations are determined by fitting the fluorescence intensity distribution with a theoretical Gaussian. We also show that BaLM works with a spectrum of fluorescent molecules in the same sample. Thus, BaLM extends single molecule-based superresolution localization to samples labeled with multiple conventional fluorescent probes.

Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less

Pump-probe nonlinear phase dispersion spectroscopy.

PubMed

Robles, Francisco E; Samineni, Prathyush; Wilson, Jesse W; Warren, Warren S

2013-04-22

Pump-probe microscopy is an imaging technique that delivers molecular contrast of pigmented samples. Here, we introduce pump-probe nonlinear phase dispersion spectroscopy (PP-NLDS), a method that leverages pump-probe microscopy and spectral-domain interferometry to ascertain information from dispersive and resonant nonlinear effects. PP-NLDS extends the information content to four dimensions (phase, amplitude, wavelength, and pump-probe time-delay) that yield unique insight into a wider range of nonlinear interactions compared to conventional methods. This results in the ability to provide highly specific molecular contrast of pigmented and non-pigmented samples. A theoretical framework is described, and experimental results and simulations illustrate the potential of this method. Implications for biomedical imaging are discussed.

Pump-probe nonlinear phase dispersion spectroscopy

PubMed Central

Robles, Francisco E.; Samineni, Prathyush; Wilson, Jesse W.; Warren, Warren S.

2013-01-01

Pump-probe microscopy is an imaging technique that delivers molecular contrast of pigmented samples. Here, we introduce pump-probe nonlinear phase dispersion spectroscopy (PP-NLDS), a method that leverages pump-probe microscopy and spectral-domain interferometry to ascertain information from dispersive and resonant nonlinear effects. PP-NLDS extends the information content to four dimensions (phase, amplitude, wavelength, and pump-probe time-delay) that yield unique insight into a wider range of nonlinear interactions compared to conventional methods. This results in the ability to provide highly specific molecular contrast of pigmented and non-pigmented samples. A theoretical framework is described, and experimental results and simulations illustrate the potential of this method. Implications for biomedical imaging are discussed. PMID:23609646

Alternative techniques in root canal debridement

NASA Astrophysics Data System (ADS)

Luca, Ruxandra; Todea, Carmen; Bǎlǎbuc, Cosmin; Nica, Luminita; Armani, Giacomo; Locovei, Cosmin

2014-01-01

Studies have demonstrated that conventional chemo-mechanical preparation is limited regarding the decontamination of the endodontic space, which is why alternative techniques such as laser radiation have their importance in the modern endodontic treatment. The present study aims to assess the possibility of improving the debridement of the root canals by removing smear layer using Er: YAG laser radiation. We used 18 extracted teeth, which were subjected to the same initial protocol and then divided into 5 study groups: the control group has not been treated with laser; the other 4 groups were exposed to laser radiation using two different geometries peaks of quartz and two energy levels. Scanning electronic microscopy revealed an increased efficiency in the debridement of all interested areas when using PIPS and XPulse tips at proper energy. In the two groups treated with inferior laser energy, the debridement didn't prove to be superior to the conventional treatment.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching

PubMed Central

Gerbich, Therese M.; Rana, Kishan; Suzuki, Aussie; Schaefer, Kristina N.; Heppert, Jennifer K.; Boothby, Thomas C.; Allbritton, Nancy L.; Gladfelter, Amy S.; Maddox, Amy S.

2018-01-01

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. PMID:29490939

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Improving multiphoton STED nanoscopy with separation of photons by LIfetime Tuning (SPLIT)

NASA Astrophysics Data System (ADS)

Coto Hernández, Iván.; Lanzano, Luca; Castello, Marco; Jowett, Nate; Tortarolo, Giorgio; Diaspro, Alberto; Vicidomini, Giuseppe

2018-02-01

Stimulated emission depletion (STED) microscopy is a powerful bio-imaging technique since it provides molecular spatial resolution whilst preserving the most important assets of fluorescence microscopy. When combined with twophoton excitation (2PE) microscopy (2PE-STED), the sub-diffraction imaging ability of STED microscopy can be achieved also on thick biological samples. The most straightforward implementation of 2PE-STED microscopy is obtained by introducing a STED beam operating in continuous wave (CW) into a conventional Ti:Sapphire based 2PE microscope (2PE-CW-STED). In this implementation, an effective resolution enhancement is mainly obtained implementing a time-gated detection scheme, which however can drastically reduce the signal-to-noise/background ratio of the final image. Herein, we combine the lifetime tuning (SPLIT) approach with 2PE-CW-STED to overcome this limitation. The SPLIT approach is employed to discard fluorescence photons lacking super-resolution information, by means of a pixel-by-pixel phasor approach. Combining the SPLIT approach with image deconvolution further optimizes the signal-to-noise/background ratio.

High-Resolution Microscopy-Coil MR Imaging of Skin Tumors: Techniques and Novel Clinical Applications.

PubMed

Budak, Matthew J; Weir-McCall, Jonathan R; Yeap, Phey M; White, Richard D; Waugh, Shelley A; Sudarshan, Thiru A P; Zealley, Ian A

2015-01-01

High-resolution magnetic resonance (MR) imaging performed with a microscopy coil is a robust radiologic tool for the evaluation of skin lesions. Microscopy-coil MR imaging uses a small surface coil and a 1.5-T or higher MR imaging system. Simple T1- and T2-weighted imaging protocols can be implemented to yield high-quality, high-spatial-resolution images that provide an excellent depiction of dermal anatomy. The primary application of microscopy-coil MR imaging is to delineate the deep margins of skin tumors, thereby providing a preoperative road map for dermatologic surgeons. This information is particularly useful for surgeons who perform Mohs micrographic surgery and in cases of nasofacial neoplasms, where the underlying anatomy is complex. Basal cell carcinoma is the most common nonmelanocytic skin tumor and has a predilection to manifest on the face, where it can be challenging to achieve complete surgical excision while preserving the cosmetic dignity of the patient. Microscopy-coil MR imaging provides dermatologic surgeons with valuable preoperative anatomic information that is not available at conventional clinical examination. ©RSNA, 2015.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

PubMed

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

2016-03-15

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

PubMed

Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

2017-10-20

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy.

PubMed

Carson, Johnny L

2014-09-11

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Four-Dimensional Ultrafast Electron Microscopy: Insights into an Emerging Technique.

PubMed

Adhikari, Aniruddha; Eliason, Jeffrey K; Sun, Jingya; Bose, Riya; Flannigan, David J; Mohammed, Omar F

2017-01-11

Four-dimensional ultrafast electron microscopy (4D-UEM) is a novel analytical technique that aims to fulfill the long-held dream of researchers to investigate materials at extremely short spatial and temporal resolutions by integrating the excellent spatial resolution of electron microscopes with the temporal resolution of ultrafast femtosecond laser-based spectroscopy. The ingenious use of pulsed photoelectrons to probe surfaces and volumes of materials enables time-resolved snapshots of the dynamics to be captured in a way hitherto impossible by other conventional techniques. The flexibility of 4D-UEM lies in the fact that it can be used in both the scanning (S-UEM) and transmission (UEM) modes depending upon the type of electron microscope involved. While UEM can be employed to monitor elementary structural changes and phase transitions in samples using real-space mapping, diffraction, electron energy-loss spectroscopy, and tomography, S-UEM is well suited to map ultrafast dynamical events on materials surfaces in space and time. This review provides an overview of the unique features that distinguish these techniques and also illustrates the applications of both S-UEM and UEM to a multitude of problems relevant to materials science and chemistry.

Contact resonance atomic force microscopy imaging in air and water using photothermal excitation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kocun, Marta; Labuda, Aleksander; Gannepalli, Anil

2015-08-15

Contact Resonance Force Microscopy (CR-FM) is a leading atomic force microscopy technique for measuring viscoelastic nano-mechanical properties. Conventional piezo-excited CR-FM measurements have been limited to imaging in air, since the “forest of peaks” frequency response associated with acoustic excitation methods effectively masks the true cantilever resonance. Using photothermal excitation results in clean contact, resonance spectra that closely match the ideal frequency response of the cantilever, allowing unambiguous and simple resonance frequency and quality factor measurements in air and liquids alike. This extends the capabilities of CR-FM to biologically relevant and other soft samples in liquid environments. We demonstrate CR-FM inmore » air and water on both stiff silicon/titanium samples and softer polystyrene-polyethylene-polypropylene polymer samples with the quantitative moduli having very good agreement between expected and measured values.« less

A user's guide to localization-based super-resolution fluorescence imaging.

PubMed

Dempsey, Graham T

2013-01-01

Advances in far-field fluorescence microscopy over the past decade have led to the development of super-resolution imaging techniques that provide more than an order of magnitude improvement in spatial resolution compared to conventional light microscopy. One such approach, called Stochastic Optical Reconstruction Microscopy (STORM) uses the sequential, nanometer-scale localization of individual fluorophores to reconstruct a high-resolution image of a structure of interest. This is an attractive method for biological investigation at the nanoscale due to its relative simplicity, both conceptually and practically in the laboratory. Like most research tools, however, the devil is in the details. The aim of this chapter is to serve as a guide for applying STORM to the study of biological samples. This chapter will discuss considerations for choosing a photoswitchable fluorescent probe, preparing a sample, selecting hardware for data acquisition, and collecting and analyzing data for image reconstruction. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

PubMed

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

Special raster scanning for reduction of charging effects in scanning electron microscopy.

PubMed

Suzuki, Kazuhiko; Oho, Eisaku

2014-01-01

A special raster scanning (SRS) method for reduction of charging effects is developed for the field of SEM. Both a conventional fast scan (horizontal direction) and an unusual scan (vertical direction) are adopted for acquiring raw data consisting of many sub-images. These data are converted to a proper SEM image using digital image processing techniques. About sharpness of the image and reduction of charging effects, the SRS is compared with the conventional fast scan (with frame-averaging) and the conventional slow scan. Experimental results show the effectiveness of SRS images. By a successful combination of the proposed scanning method and low accelerating voltage (LV)-SEMs, it is expected that higher-quality SEM images can be more easily acquired by the considerable reduction of charging effects, while maintaining the resolution. © 2013 Wiley Periodicals, Inc.

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

NASA Astrophysics Data System (ADS)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

Three-dimensional molecular mapping of a multiple emulsion by means of CARS microscopy.

PubMed

Meyer, Tobias; Akimov, Denis; Tarcea, Nicolae; Chatzipapadopoulos, Susana; Muschiolik, Gerald; Kobow, Jens; Schmitt, Michael; Popp, Jürgen

2008-02-07

Multiple emulsions consisting of water droplets dispersed in an oil phase containing emulsifier which is emulsified in an outer water phase (W/O/W) are of great interest in pharmacology for developing new drugs, in the nutrition sciences for designing functional food, and in biology as model systems for cell organelles such as liposomes. In the food industry multiple emulsions with high sugar content in the aqueous phase can be used for the production of sweets, because the high sugar content prevents deterioration. However, for these emulsions the refractive indexes of oil and aqueous phase are very similar. This seriously impedes the analysis of these emulsions, e.g., for process monitoring, because microscopic techniques based on transmission or reflection do not provide sufficient contrast. We have characterized the inner dispersed phase of concentrated W/O/W emulsions with the same refractive index of the three phases by micro Raman spectroscopy and investigated the composition and molecular distribution in water-oil-water emulsions by means of three-dimensional laser scanning CARS (coherent anti-Stokes Raman scattering) microscopy. CARS microscopy has been used to study water droplets dispersed in oil droplets at different Raman resonances to visualize different molecular species. Water droplets with a diameter of about 700 nm could clearly be visualized. The advantages of CARS microscopy for studying this particular system are emphasized by comparing this microscopic technique with conventional confocal reflection and transmission microscopies.

NMR Microscopy - Micron-Level Resolution.

NASA Astrophysics Data System (ADS)

Kwok, Wing-Chi Edmund

1990-01-01

Nuclear Magnetic Resonance Imaging (MRI) has been developed into a powerful and widely used diagnostic tool since the invention of techniques using linear magnetic field gradients in 1973. The variety of imaging contrasts obtainable in MRI, such as spin density, relaxation times and flow rate, gives MRI a significant advantage over other imaging techniques. For common diagnostic applications, image resolutions have been in the order of millimeters with slice thicknesses in centimeters. For many research applications, however, resolutions in the order of tens of microns or smaller are needed. NMR Imaging in these high resolution disciplines is known as NMR microscopy. Compared with conventional microscopy, NMR microscopy has the advantage of being non-invasive and non-destructive. The major obstacles of NMR microscopy are low signal-to-noise ratio and effects due to spin diffusion. To overcome these difficulties, more sensitive RF probes and very high magnetic field gradients have to be used. The most effective way to increase sensitivity is to build smaller probes. Microscope probes of different designs have been built and evaluated. Magnetic field gradient coils that can produce linear field gradients up to 450 Gauss/cm were also assembled. In addition, since microscope probes often employ remote capacitors for RF tuning, the associated signal loss in the transmission line was studied. Imaging experiments have been carried out in a 2.1 Tesla small bore superconducting magnet using the typical two-dimensional spin warp imaging technique. Images have been acquired for both biological and non-biological samples. The highest resolution was obtained in an image of a nerve bundle from the spinal cord of a racoon and has an in-plane resolution of 4 microns. These experiments have demonstrated the potential application of NMR microscopy to pathological research, nervous system study and non -destructive testings of materials. One way to further improve NMR microscopy is to implement a higher static magnetic field which will increase signal strength. In the future, NMR microscopy should prove to be useful in the studies of cell linings, T1 & T2 relaxation mechanisms and NMR contrast agents.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

The actin cytoskeleton in whole mount preparations and sections.

PubMed

Resch, Guenter P; Urban, Edit; Jacob, Sonja

2010-01-01

In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

[Cadmium citotoxicity in mice hepatocytes and implications on tropical environments].

PubMed

Marcano, Letty; Faría, Clarisa de R; Carruyo, Ingrid; Montiel, Xiomara

2006-06-01

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

In vivo functional photoacoustic microscopy of cutaneous microvasculature in human skin.

PubMed

Favazza, Christopher P; Cornelius, Lynn A; Wang, Lihong V

2011-02-01

Microcirculation is an important component of the cardiovascular system and can be used to assess systemic cardiovascular health. Numerous studies have investigated cutaneous microcirculation as an indicator of cardiovascular related diseases. Such research has shown promising results; however, there are many limitations regarding the employed measurement techniques, such as poor depth and spatial resolution and measurement versatility. Here we show the results of functional cutaneous microvascular experiments measured with photoacoustic microscopy, which provides high spatial resolution and multiparameter measurements. In a set of experiments, microvascular networks located in the palms of volunteers were perturbed by periodic ischemic events, and the subsequent hemodynamic response to the stimulus was recorded. Results indicate that during periods of arterial occlusion, the relative oxygen saturation of the capillary vessels decreased below resting levels, and temporarily increased above resting levels immediately following the occlusion. Furthermore, a hyperemic reaction to the occlusions was measured, and the observation agreed well with similar measurements using more conventional imaging techniques. Due to its exceptional capability to functionally image vascular networks with high spatial resolution, photoacoustic microscopy could be a beneficial biomedical tool to assess microvascular functioning and applied to patients with diseases that affect cardiovascular health. © 2011 Society of Photo-Optical Instrumentation Engineers.

A high-throughput comparative characterization of laser-induced soft tissue damage using 3D digital microscopy.

PubMed

Das, Debobrato; Reed, Stephanie; Klokkevold, Perry R; Wu, Benjamin M

2013-02-01

3D digital microscopy was used to develop a rapid alternative approach to quantify the effects of specific laser parameters on soft tissue ablation and charring in vitro without the use of conventional tissue processing techniques. Two diode lasers operating at 810 and 980 nm wavelengths were used to ablate three tissue types (bovine liver, turkey breast, and bovine muscle) at varying laser power (0.3, 1.0, and 2.0 W) and velocities (1-50 mm/s). Spectrophotometric analyses were performed on each tissue to determine tissue-specific absorption coefficients and were considered in creating wavelength-dependent energy attenuation models to evaluate minimum heat of tissue ablations. 3D surface contour profiles characterizing tissue damage revealed that ablation depth and tissue charring increased with laser power and decreased with lateral velocity independent of wavelength and tissue type. While bovine liver ablation and charring were statistically higher at 810 than 980 nm (p < 0.05), turkey breast and bovine muscle ablated and charred more at 980 than 810 nm (p < 0.05). Spectrophotometric analysis revealed that bovine liver tissue had a greater tissue-specific absorption coefficient at 810 than 980 nm, while turkey breast and bovine muscle had a larger absorption coefficient at 980 nm (p < 0.05). This rapid 3D microscopic analysis of robot-driven laser ablation yielded highly reproducible data that supported well-defined trends related to laser-tissue interactions and enabled high throughput characterization of many laser-tissue permutations. Since 3D microscopy quantifies entire lesions without altering the tissue specimens, conventional and immunohistologic techniques can be used, if desired, to further interrogate specific sections of the digitized lesions.

Light-neuron interactions: key to understanding the brain

NASA Astrophysics Data System (ADS)

Go, Mary Ann; Daria, Vincent R.

2017-02-01

In recent years, advances in light-based technology have driven an ongoing optical revolution in neuroscience. Synergistic technologies in laser microscopy, molecular biology, organic and synthetic chemistry, genetic engineering and materials science have allowed light to overcome the limitations of and to replace many conventional tools used by physiologists to record from and to manipulate single cells or whole cellular networks. Here we review the different optical techniques for stimulating neurons, influencing neuronal growth, manipulating neuronal structures and neurosurgery.

Microstructural Analysis of Ti-6Al-4V Components Made by Electron Beam Additive Manufacturing

NASA Astrophysics Data System (ADS)

Coleman, Rashadd L.

Electron Beam Additive Manufacturing (EBAM) is a relatively new additive manufacturing (AM) technology that uses a high-energy electron beam to melt and fuse powders to build full-density parts in a layer by layer fashion. EBAM can fabricate metallic components, particularly, of complex shapes, in an efficient and cost-effective manner compared to conventional manufacturing means. EBAM is an enabling technology for rapid manufacturing (RM) of metallic components, and thus, can efficiently integrate the design and manufacturing of aerospace components. However, EBAM for aerospace-related applications remain limited because the effect of the EBAM process on part characteristics is not fully understood. In this study, various techniques including microhardness, optical microscopy (OM), X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and electron backscatter diffraction (EBSD) were used to characterize Ti-6Al-4V components processed using EBAM. The results were compared to Ti-6Al-4V components processed using conventional techniques. In this study it is shown that EBAM built Ti-64 components have increased hardness, elastic modulus, and yield strength compared to wrought Ti-6Al-4V. Further, it is also shown in this study that the horizontal build EBAM Ti-6Al-4V has increased hardness, elastic modulus, and yield strength compared to vertical build EBAM due to a preferential growth of the beta phase.

Optical diffraction tomography with fully and partially coherent illumination in high numerical aperture label-free microscopy [Invited].

PubMed

Soto, Juan M; Rodrigo, José A; Alieva, Tatiana

2018-01-01

Quantitative label-free imaging is an important tool for the study of living microorganisms that, during the last decade, has attracted wide attention from the optical community. Optical diffraction tomography (ODT) is probably the most relevant technique for quantitative label-free 3D imaging applied in wide-field microscopy in the visible range. The ODT is usually performed using spatially coherent light illumination and specially designed holographic microscopes. Nevertheless, the ODT is also compatible with partially coherent illumination and can be realized in conventional wide-field microscopes by applying refocusing techniques, as it has been recently demonstrated. Here, we compare these two ODT modalities, underlining their pros and cons and discussing the optical setups for their implementation. In particular, we pay special attention to a system that is compatible with a conventional wide-field microscope that can be used for both ODT modalities. It consists of two easily attachable modules: the first for sample illumination engineering based on digital light processing technology; the other for focus scanning by using an electrically driven tunable lens. This hardware allows for a programmable selection of the wavelength and the illumination design, and provides fast data acquisition as well. Its performance is experimentally demonstrated in the case of ODT with partially coherent illumination providing speckle-free 3D quantitative imaging.

Resinless section electron microscopy reveals the yeast cytoskeleton.

PubMed

Penman, J; Penman, S

1997-04-15

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

PubMed Central

Sarder, Pinaki; Yazdanfar, Siavash; Akers, Walter J.; Tang, Rui; Sudlow, Gail P.; Egbulefu, Christopher

2013-01-01

Abstract. The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100  μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500  μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms. PMID:24150231

Resinless section electron microscopy reveals the yeast cytoskeleton

PubMed Central

Penman, Joshua; Penman, Sheldon

1997-01-01

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture. PMID:9108046

Rethinking cell structure.

PubMed Central

Penman, S

1995-01-01

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7777493

In situ X-ray ptychography imaging of high-temperature CO2 acceptor particle agglomerates

NASA Astrophysics Data System (ADS)

Høydalsvik, Kristin; Bø Fløystad, Jostein; Zhao, Tiejun; Esmaeili, Morteza; Diaz, Ana; Andreasen, Jens W.; Mathiesen, Ragnvald H.; Rønning, Magnus; Breiby, Dag W.

2014-06-01

Imaging nanoparticles under relevant reaction conditions of high temperature and gas pressure is difficult because conventional imaging techniques, like transmission electron microscopy, cannot be used. Here we demonstrate that the coherent diffractive imaging technique of X-ray ptychography can be used for in situ phase contrast imaging in structure studies at atmospheric pressure and elevated temperatures. Lithium zirconate, a candidate CO2 capture material, was studied at a pressure of one atmosphere in air and in CO2, at temperatures exceeding 600 °C. Images with a spatial resolution better than 200 nm were retrieved, and possibilities for improving the experiment are described.

3D light scanning macrography.

PubMed

Huber, D; Keller, M; Robert, D

2001-08-01

The technique of 3D light scanning macrography permits the non-invasive surface scanning of small specimens at magnifications up to 200x. Obviating both the problem of limited depth of field inherent to conventional close-up macrophotography and the metallic coating required by scanning electron microscopy, 3D light scanning macrography provides three-dimensional digital images of intact specimens without the loss of colour, texture and transparency information. This newly developed technique offers a versatile, portable and cost-efficient method for the non-invasive digital and photographic documentation of small objects. Computer controlled device operation and digital image acquisition facilitate fast and accurate quantitative morphometric investigations, and the technique offers a broad field of research and educational applications in biological, medical and materials sciences.

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

Interface formation in monolayer graphene-boron nitride heterostructures.

PubMed

Sutter, P; Cortes, R; Lahiri, J; Sutter, E

2012-09-12

The ability to control the formation of interfaces between different materials has become one of the foundations of modern materials science. With the advent of two-dimensional (2D) crystals, low-dimensional equivalents of conventional interfaces can be envisioned: line boundaries separating different materials integrated in a single 2D sheet. Graphene and hexagonal boron nitride offer an attractive system from which to build such 2D heterostructures. They are isostructural, nearly lattice-matched, and isoelectronic, yet their different band structures promise interesting functional properties arising from their integration. Here, we use a combination of in situ microscopy techniques to study the growth and interface formation of monolayer graphene-boron nitride heterostructures on ruthenium. In a sequential chemical vapor deposition process, boron nitride grows preferentially at the edges of existing monolayer graphene domains, which can be exploited for synthesizing continuous 2D membranes of graphene embedded in boron nitride. High-temperature growth leads to intermixing near the interface, similar to interfacial alloying in conventional heterostructures. Using real-time microscopy, we identify processes that eliminate this intermixing and thus pave the way to graphene-boron nitride heterostructures with atomically sharp interfaces.

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

PubMed Central

Li, Yiwen; Song, Ying; Zhao, Lian; Gaidosh, Gabriel; Laties, Alan M; Wen, Rong

2009-01-01

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis. PMID:18846097

[Small-cell anaplastic neuroendocrine carcinoma of the rectum].

PubMed

Molas, G; Bougis-de-Brux, M A; Potet, F

1987-12-01

A pediculed tumor of the rectum was discovered in a 63 years old man. Within the tumor adenomatous dysplastic proliferation was associated with a neuroendocrine small-cell anaplastic carcinoma. The neuroendocrine nature of the tumor was suspected on conventional optic microscopy and confirmed by a positive Grimelius technique. Specific typical granules were also found on electron microscopy. Immunohistochemical techniques using neurospecific enolase were also positive. Carcinomatous invasion was limited to the submucosa, but the surgical specimen showed that one lymph node was metastatic. Three months later, hepatic metastasis was suspected on physical examination and the patient died of hepatic failure ten months after the discovery of the tumor. Twenty-two similar cases were found in the literature: of these five cases were associated with benign adenomatous lesions. In all cases the patients died of early metastatic diffusion. This tumor raises the problems of diagnosis, terminology, classification and therapy: only aggressive chemotherapy, similar to that applied to the same type of carcinoma in the respiratory tract might improve prognosis.

Holographic quantitative imaging of sample hidden by turbid medium or occluding objects

NASA Astrophysics Data System (ADS)

Bianco, V.; Miccio, L.; Merola, F.; Memmolo, P.; Gennari, O.; Paturzo, Melania; Netti, P. A.; Ferraro, P.

2015-03-01

Digital Holography (DH) numerical procedures have been developed to allow imaging through turbid media. A fluid is considered turbid when dispersed particles provoke strong light scattering, thus destroying the image formation by any standard optical system. Here we show that sharp amplitude imaging and phase-contrast mapping of object hidden behind turbid medium and/or occluding objects are possible in harsh noise conditions and with a large field-of view by Multi-Look DH microscopy. In particular, it will be shown that both amplitude imaging and phase-contrast mapping of cells hidden behind a flow of Red Blood Cells can be obtained. This allows, in a noninvasive way, the quantitative evaluation of living processes in Lab on Chip platforms where conventional microscopy techniques fail. The combination of this technique with endoscopic imaging can pave the way for the holographic blood vessel inspection, e.g. to look for settled cholesterol plaques as well as blood clots for a rapid diagnostics of blood diseases.

Electrostatic nanolithography in polymer materials: an alternative technique for nanostructures formation

NASA Astrophysics Data System (ADS)

Lyuksyutov, Sergei F.; Paramonov, Pavel B.; Sigalov, Grigori; Vaia, Richard A.; Juhl, Shane; Sancaktar, Erol

2003-10-01

The combination of localized softening attolitres (10^2 -10^4) of polymer film by Jule heating, extremely non-uniform electric field gradients to polarize and manipulate the soften polymer, and single step technique using conventional atomic force microscopy (AFM), establishes a new paradigm for nanolithography in a broad class of polymer materials allowing rapid (order of milliseconds) creation of raised and depressed nanostructures without external heating of a polymer film of AFM tip-film contact [1]. In this work we present recent studies of AFM-assisted electrostatic nanolithography (AFMEN) such as amplitude-modulated AFMEN, and the humidity influence on nanostructures formation during contact mode AFMEN. It has been shown that the aspect ratio of nanostructures grows on the order of magnitude (0.2), while the lateral dimensions of nanodots decreases down to 10-15 nm. [1] S.F. Lyuksyutov, R.A. Vaia, P.B. Paramonov, S. Juhl, L. Waterhouse, R.M. Ralich, G. Sigalov, and E. Sancaktar, "Electrostatic nanolithography in polymers using atomic force microscopy," Nature Materials 2, 468-472 (2003)

Hemoglobin protein hollow shells fabricated through covalent layer-by-layer technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duan Li; He Qiang; Max Planck Institute of Colloids and Interfaces, Golm/Potsdam D-14476

2007-03-09

Hemoglobin (Hb) protein microcapsules held together by cross-linker, glutaraldehyde (GA), were successfully fabricated by covalent layer-by-layer (LbL) technique. The Schiff base reaction occurred on the colloid templates between the aldehyde groups of GA and free amino sites of Hb results in the formation of GA/Hb microcapsules after the removal of the templates. The structure of obtained monodisperse protein microcapsule was characterized by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). The UV-Vis spectra measurements demonstrate the existence of Hb in the assembled capsules. Cyclic voltammetry (CV) and potential-controlled amperometric measurements (I-t curve) confirm that hemoglobin microcapsules after fabricationmore » remain their heme electroactivity. Moreover, direct electron transfer process from protein to electrode surface was performed to detect the heme electrochemistry without using any mediator or promoter. The experiments of fluorescence recovery after photobleaching (FRAP) by CLSM demonstrate that the hemoglobin protein microcapsules have an improved permeability comparing to the conventional polyelectrolyte microcapsules.« less

Structured illumination microscopy for dual-modality 3D sub-diffraction resolution fluorescence and refractive-index reconstruction

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Though structured illumination (SI) microscopy is a popular imaging technique conventionally associated with fluorescent super-resolution, recent works have suggested its applicability towards sub-diffraction resolution coherent imaging with quantitative endogenous biological contrast. Here, we demonstrate that SI can efficiently integrate together the principles of fluorescent super-resolution and coherent synthetic aperture to achieve 3D dual-modality sub-diffraction resolution, fluorescence and refractive-index (RI) visualizations of biological samples. We experimentally demonstrate this framework by introducing a SI microscope capable of 3D sub-diffraction resolution fluorescence and RI imaging, and verify its biological visualization capabilities by experimentally reconstructing 3D RI/fluorescence visualizations of fluorescent calibration microspheres as well as alveolar basal epithelial adenocarcinoma (A549) and human colorectal adenocarcinmoa (HT-29) cells, fluorescently stained for F-actin. This demonstration may suggest SI as an especially promising imaging technique to enable future biological studies that explore synergistically operating biophysical/biochemical and molecular mechanisms at sub-diffraction resolutions. PMID:29296504

Scanning electron microscopy comparison of the cleaning efficacy of a root canal system by Nd:YAG laser and rotary instruments.

PubMed

Samiei, Mohammad; Pakdel, Seyyed Mahdi Vahid; Rikhtegaran, Sahand; Shakoei, Sahar; Ebrahimpour, Delaram; Taghavi, Pedram

2014-08-01

This study evaluated the cleaning efficacy of a root canal system by Nd:YAG laser and rotary instruments. Sixty single-rooted human teeth were divided into four experimental groups (n=15). In the first group the teeth were prepared with a step-back technique using conventional K-files. In the second and third groups, tooth preparation was carried out using Nd:YAG laser and rotary NiTi instruments, respectively. Teeth in the fourth group were prepared by combined laser and rotary methods. The smear layer remaining on canal walls was then assessed by scanning electron microscopy in the coronal, middle, and apical portions. The comparison of smear layer removal efficacy between groups was carried out by Kruskal-Wallis and Mann-Whitney U tests. The mean grades of smear layer removal in rotary-laser, rotary, laser and step-back techniques were 1.34 ± 0.18, 2.2 ± 0.28, 1.91 ± 0.25, and 2.42 ± 0.19, respectively. On the whole, differences between rotary-laser and rotary groups, step-back, and the three other techniques (rotary, laser, and rotary-laser) were significant at p=0.034. Based on the findings of this study, the cleaning efficacy of rotary, laser, and rotary-laser techniques were better than the step-back technique and the combined laser and rotary technique was the most efficient method.

Evaluation of Microstructure and Mechanical Properties of Al-TiC Metal Matrix Composite Prepared by Conventional, Microwave and Spark Plasma Sintering Methods

PubMed Central

Ghasali, Ehsan; Fazili, Ali; Alizadeh, Masoud; Shirvanimoghaddam, Kamyar; Ebadzadeh, Touradj

2017-01-01

In this research, the mechanical properties and microstructure of Al-15 wt % TiC composite samples prepared by spark plasma, microwave, and conventional sintering were investigated. The sintering process was performed by the speak plasma sintering (SPS) technique, microwave and conventional furnaces at 400 °C, 600 °C, and 700 °C, respectively. The results showed that sintered samples by SPS have the highest relative density (99% of theoretical density), bending strength (291 ± 12 MPa), and hardness (253 ± 23 HV). The X-ray diffraction (XRD) investigations showed the formation of TiO2 from the surface layer decomposition of TiC particles. Scanning electron microscopy (SEM) micrographs demonstrated uniform distribution of reinforcement particles in all sintered samples. The SEM/EDS analysis revealed the formation of TiO2 around the porous TiC particles. PMID:29088114

Structures of Astromaterials Revealed by EBSD

NASA Technical Reports Server (NTRS)

Zolensky, M.

2018-01-01

Groups at the Johnson Space Center and the University of Tokyo have been using electron back-scattered diffraction (EBSD) to reveal the crystal structures of extraterrestrial minerals for many years. Even though we also routinely use transmission electron microscopy, synchrotron X-ray diffraction (SXRD), and conventional electron diffraction, we find that EBSD is the most powerful technique for crystal structure elucidation in many instances. In this talk I describe a few of the cases where we have found EBSD to provide crucial, unique information. See attachment.

Simultaneous measurements of bulk moduli and particle dynamics in a sheared colloidal glass

NASA Astrophysics Data System (ADS)

Massa, Michael V.; Eisenmann, Christoph; Kim, Chanjoong; Weitz, David A.

2007-03-01

We present a novel study of glassy colloidal systems, using a stress-controlled rheometer in conjunction with a confocal microscope. This experimental setup combines the measurement of bulk moduli, using conventional rheology, with the ability to track the motion of individual particles, through confocal microscopy techniques. We explore the response of the system to applied shear, by simultaneously monitoring the macroscopic relaxation and microscopic particle dynamics, under conditions from the quiescent glass to a shear-melted liquid.

In situ electrochemical digital holographic microscopy; a study of metal electrodeposition in deep eutectic solvents.

PubMed

Abbott, Andrew P; Azam, Muhammad; Ryder, Karl S; Saleem, Saima

2013-07-16

This study has shown for the first time that digital holographic microscopy (DHM) can be used as a new analytical tool in analysis of kinetic mechanism and growth during electrolytic deposition processes. Unlike many alternative established electrochemical microscopy methods such as probe microscopy, DHM is both the noninvasive and noncontact, the unique holographic imaging allows the observations and measurement to be made remotely. DHM also provides interferometric resolution (nanometer vertical scale) with a very short acquisition time. It is a surface metrology technique that enables the retrieval of information about a 3D structure from the phase contrast of a single hologram acquired using a conventional digital camera. Here DHM has been applied to investigate directly the electro-crystallization of a metal on a substrate in real time (in situ) from two deep eutectic solvent (DES) systems based on mixture of choline chloride and either urea or ethylene glycol. We show, using electrochemical DHM that the nucleation and growth of silver deposits in these systems are quite distinct and influenced strongly by the hydrogen bond donor of the DES.

Harnessing the damping properties of materials for high-speed atomic force microscopy.

PubMed

Adams, Jonathan D; Erickson, Blake W; Grossenbacher, Jonas; Brugger, Juergen; Nievergelt, Adrian; Fantner, Georg E

2016-02-01

The success of high-speed atomic force microscopy in imaging molecular motors, enzymes and microbes in liquid environments suggests that the technique could be of significant value in a variety of areas of nanotechnology. However, the majority of atomic force microscopy experiments are performed in air, and the tapping-mode detection speed of current high-speed cantilevers is an order of magnitude lower in air than in liquids. Traditional approaches to increasing the imaging rate of atomic force microscopy have involved reducing the size of the cantilever, but further reductions in size will require a fundamental change in the detection method of the microscope. Here, we show that high-speed imaging in air can instead be achieved by changing the cantilever material. We use cantilevers fabricated from polymers, which can mimic the high damping environment of liquids. With this approach, SU-8 polymer cantilevers are developed that have an imaging-in-air detection bandwidth that is 19 times faster than those of conventional cantilevers of similar size, resonance frequency and spring constant.

Richardson-Lucy deconvolution as a general tool for combining images with complementary strengths.

PubMed

Ingaramo, Maria; York, Andrew G; Hoogendoorn, Eelco; Postma, Marten; Shroff, Hari; Patterson, George H

2014-03-17

We use Richardson-Lucy (RL) deconvolution to combine multiple images of a simulated object into a single image in the context of modern fluorescence microscopy techniques. RL deconvolution can merge images with very different point-spread functions, such as in multiview light-sheet microscopes,1, 2 while preserving the best resolution information present in each image. We show that RL deconvolution is also easily applied to merge high-resolution, high-noise images with low-resolution, low-noise images, relevant when complementing conventional microscopy with localization microscopy. We also use RL deconvolution to merge images produced by different simulated illumination patterns, relevant to structured illumination microscopy (SIM)3, 4 and image scanning microscopy (ISM). The quality of our ISM reconstructions is at least as good as reconstructions using standard inversion algorithms for ISM data, but our method follows a simpler recipe that requires no mathematical insight. Finally, we apply RL deconvolution to merge a series of ten images with varying signal and resolution levels. This combination is relevant to gated stimulated-emission depletion (STED) microscopy, and shows that merges of high-quality images are possible even in cases for which a non-iterative inversion algorithm is unknown. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Virtual microscopy-The future of teaching histology in the medical curriculum?

PubMed

Paulsen, Friedrich P; Eichhorn, Michael; Bräuer, Lars

2010-12-20

Conventional continuing education in microscopic anatomy, histopathology, hematology and microbiology has hitherto been carried out using numerous sets of sectioned tissue specimens in a microscopy laboratory. In comparison, after digitalization of the sections it would be possible to access teaching specimens via virtual microscopy and the internet at any time and place. This would make it possible to put innumerable new learning scenarios into practice. The present article elucidates the advantages of virtual microscopy in histology instruction and presents a concept of how virtual microscopy could be introduced into the teaching of microscopic anatomy in several steps. Initially, the presently existing microscopic teaching specimens would be digitalized and made available on-line without restriction. In a second step, instruction would be shifted to an emphasis on virtual microscopy, utilizing all of the advantages offered by the technique. In a third step, the microscopic contents could be networked with other anatomical, radiological and clinical content on-line, thus opening new learning perspectives for students of human and dental medicine as well as those of medically related courses of study. The advantages and disadvantages of such a concept as well as some possibly arising consequences are discussed in the following. 2010 Elsevier GmbH. All rights reserved.

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

Images as tools. On visual epistemic practices in the biological sciences.

PubMed

Samuel, Nina

2013-06-01

Contemporary visual epistemic practices in the biological sciences raise new questions of how to transform an iconic data measurements into images, and how the process of an imaging technique may change the material it is 'depicting'. This case-oriented study investigates microscopic imagery, which is used by system and synthetic biologists alike. The core argument is developed around the analysis of two recent methods, developed between 2003 and 2006: localization microscopy and photo-induced cell death. Far from functioning merely as illustrations of work done by other means, images can be determined as tools for discovery in their own right and as objects of investigation. Both methods deploy different constellations of intended and unintended interactions between visual appearance and underlying biological materiality. To characterize these new ways of interaction, the article introduces the notions of 'operational images' and 'operational agency'. Despite all their novelty, operational images are still subject to conventions of seeing and depicting: Phenomena emerging with the new method of localization microscopy have to be designed according to image traditions of older, conventional fluorescence microscopy to function properly as devices for communication between physicists and biologists. The article emerged from a laboratory study based on interviews conducted with researchers from the Kirchhoff-Institute for Physics and German Cancer Research Center (DKFZ) at Bioquant, Heidelberg, in 2011. Copyright © 2013 Elsevier Ltd. All rights reserved.

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

2014-02-01

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

NASA Astrophysics Data System (ADS)

Zheng, Wei

2014-11-01

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

Mapping in vitro local material properties of intact and disrupted virions at high resolution using multi-harmonic atomic force microscopy.

PubMed

Cartagena, Alexander; Hernando-Pérez, Mercedes; Carrascosa, José L; de Pablo, Pedro J; Raman, Arvind

2013-06-07

Understanding the relationships between viral material properties (stiffness, strength, charge density, adhesion, hydration, viscosity, etc.), structure (protein sub-units, genome, surface receptors, appendages), and functions (self-assembly, stability, disassembly, infection) is of significant importance in physical virology and nanomedicine. Conventional Atomic Force Microscopy (AFM) methods have measured a single physical property such as the stiffness of the entire virus from nano-indentation at a few points which severely limits the study of structure-property-function relationships. We present an in vitro dynamic AFM technique operating in the intermittent contact regime which synthesizes anharmonic Lorentz-force excited AFM cantilevers to map quantitatively at nanometer resolution the local electro-mechanical force gradient, adhesion, and hydration layer viscosity within individual φ29 virions. Furthermore, the changes in material properties over the entire φ29 virion provoked by the local disruption of its shell are studied, providing evidence of bacteriophage depressurization. The technique significantly generalizes recent multi-harmonic theory (A. Raman, et al., Nat. Nanotechnol., 2011, 6, 809-814) and enables high-resolution in vitro quantitative mapping of multiple material properties within weakly bonded viruses and nanoparticles with complex structure that otherwise cannot be observed using standard AFM techniques.

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangum, John S.; Chan, Lisa H.; Schmidt, Ute

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In thismore » work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice.« less

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs.

PubMed

Mangum, John S; Chan, Lisa H; Schmidt, Ute; Garten, Lauren M; Ginley, David S; Gorman, Brian P

2018-05-01

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In this work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice. Copyright © 2018 Elsevier B.V. All rights reserved.

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs

DOE PAGES

Mangum, John S.; Chan, Lisa H.; Schmidt, Ute; ...

2018-02-23

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In thismore » work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice.« less

Influence of different restorative techniques on marginal seal of class II composite restorations

PubMed Central

RODRIGUES JUNIOR, Sinval Adalberto; PIN, Lúcio Fernando da Silva; MACHADO, Giovanna; DELLA BONA, Álvaro; DEMARCO, Flávio Fernando

2010-01-01

Objective To evaluate the gingival marginal seal in class II composite restorations using different restorative techniques. Material and Methods Class II box cavities were prepared in both proximal faces of 32 sound human third molars with gingival margins located in either enamel or dentin/cementum. Restorations were performed as follows: G1 (control): composite, conventional light curing technique; G2: composite, soft-start technique; G3: amalgam/composite association (amalcomp); and G4: resin-modified glass ionomer cement/ composite, open sandwich technique. The restored specimens were thermocycled. Epoxy resin replicas were made and coated for scanning electron microscopy examination. For microleakage evaluation, teeth were coated with nail polish and immersed in dye solution. Teeth were cut in 3 slices and dye penetration was recorded (mm), digitized and analyzed with Image Tool software. Microleakage data were analyzed statistically by non-parametric Kruskal-Wallis and Mann-Whitney tests. Results Leakage in enamel was lower than in dentin (p<0.001). G2 exhibited the lowest leakage values (p<0.05) in enamel margins, with no differences between the other groups. In dentin margins, groups G1 and G2 had similar behavior and both showed less leakage (p<0.05) than groups G3 and G4. SEM micrographs revealed different marginal adaptation patterns for the different techniques and for the different substrates. Conclusion The soft-start technique showed no leakage in enamel margins and produced similar values to those of the conventional (control) technique for dentin margins. PMID:20379680

Electron energy loss spectroscopy techniques for the study of microbial chromium(VI) reduction

NASA Technical Reports Server (NTRS)

Daulton, Tyrone L.; Little, Brenda J.; Lowe, Kristine; Jones-Meehan, Joanne

2002-01-01

Electron energy loss spectroscopy (EELS) techniques were used to determine oxidation state, at high spatial resolution, of chromium associated with the metal-reducing bacteria, Shewanella oneidensis, in anaerobic cultures containing Cr(VI)O4(2-). These techniques were applied to fixed cells examined in thin section by conventional transmission electron microscopy (TEM) as well as unfixed, hydrated bacteria examined by environmental cell (EC)-TEM. Two distinct populations of bacteria were observed by TEM: bacteria exhibiting low image contrast and bacteria exhibiting high contrast in their cell membrane (or boundary) structure which was often encrusted with high-contrast precipitates. Measurements by EELS demonstrated that cell boundaries became saturated with low concentrations of Cr and the precipitates encrusting bacterial cells contained a reduced form of Cr in oxidation state + 3 or lower.

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

In vivo functional photoacoustic microscopy of cutaneous microvasculature in human skin

PubMed Central

Favazza, Christopher P.; Cornelius, Lynn A.; Wang, Lihong V.

2011-01-01

Microcirculation is an important component of the cardiovascular system and can be used to assess systemic cardiovascular health. Numerous studies have investigated cutaneous microcirculation as an indicator of cardiovascular related diseases. Such research has shown promising results; however, there are many limitations regarding the employed measurement techniques, such as poor depth and spatial resolution and measurement versatility. Here we show the results of functional cutaneous microvascular experiments measured with photoacoustic microscopy, which provides high spatial resolution and multiparameter measurements. In a set of experiments, microvascular networks located in the palms of volunteers were perturbed by periodic ischemic events, and the subsequent hemodynamic response to the stimulus was recorded. Results indicate that during periods of arterial occlusion, the relative oxygen saturation of the capillary vessels decreased below resting levels, and temporarily increased above resting levels immediately following the occlusion. Furthermore, a hyperemic reaction to the occlusions was measured, and the observation agreed well with similar measurements using more conventional imaging techniques. Due to its exceptional capability to functionally image vascular networks with high spatial resolution, photoacoustic microscopy could be a beneficial biomedical tool to assess microvascular functioning and applied to patients with diseases that affect cardiovascular health. © 2011 Society of Photo-Optical Instrumentation Engineers. PMID:21361688

In situ X-ray ptychography imaging of high-temperature CO{sub 2} acceptor particle agglomerates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Høydalsvik, Kristin; Bø Fløystad, Jostein; Esmaeili, Morteza

2014-06-16

Imaging nanoparticles under relevant reaction conditions of high temperature and gas pressure is difficult because conventional imaging techniques, like transmission electron microscopy, cannot be used. Here we demonstrate that the coherent diffractive imaging technique of X-ray ptychography can be used for in situ phase contrast imaging in structure studies at atmospheric pressure and elevated temperatures. Lithium zirconate, a candidate CO{sub 2} capture material, was studied at a pressure of one atmosphere in air and in CO{sub 2}, at temperatures exceeding 600 °C. Images with a spatial resolution better than 200 nm were retrieved, and possibilities for improving the experiment are described.

Pump-probe spectroscopy in organic semiconductors: monitoring fundamental processes of relevance in optoelectronics.

PubMed

Cabanillas-Gonzalez, Juan; Grancini, Giulia; Lanzani, Guglielmo

2011-12-08

In this review we highlight the contribution of pump-probe spectroscopy to understand elementary processes taking place in organic based optoelectronic devices. The techniques described in this article span from conventional pump-probe spectroscopy to electromodulated pump-probe and the state-of-the-art confocal pump-probe microscopy. The article is structured according to three fundamental processes (optical gain, charge photogeneration and charge transport) and the contribution of these techniques on them. The combination of these tools opens up new perspectives for assessing the role of short-lived excited states on processes lying underneath organic device operation. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Practical Framework for an Electron Beam Induced Current Technique Based on a Numerical Optimization Approach

NASA Astrophysics Data System (ADS)

Yamaguchi, Hideshi; Soeda, Takeshi

2015-03-01

A practical framework for an electron beam induced current (EBIC) technique has been established for conductive materials based on a numerical optimization approach. Although the conventional EBIC technique is useful for evaluating the distributions of dopants or crystal defects in semiconductor transistors, issues related to the reproducibility and quantitative capability of measurements using this technique persist. For instance, it is difficult to acquire high-quality EBIC images throughout continuous tests due to variation in operator skill or test environment. Recently, due to the evaluation of EBIC equipment performance and the numerical optimization of equipment items, the constant acquisition of high contrast images has become possible, improving the reproducibility as well as yield regardless of operator skill or test environment. The technique proposed herein is even more sensitive and quantitative than scanning probe microscopy, an imaging technique that can possibly damage the sample. The new technique is expected to benefit the electrical evaluation of fragile or soft materials along with LSI materials.

Simultaneous recording of electrical activity and the underlying ionic currents in NG108-15 cells cultured on gold substrate.

PubMed

Acosta-García, Ma Cristina; Morales-Reyes, Israel; Jiménez-Anguiano, Anabel; Batina, Nikola; Castellanos, N P; Godínez-Fernández, R

2018-02-01

This paper shows the simultaneous recording of electrical activity and the underlying ionic currents by using a gold substrate to culture NG108-15 cells. Cells grown on two different substrates (plastic Petri dishes and gold substrates) were characterized quantitatively through scanning electron microscopy (SEM) as well as qualitatively by optical and atomic force microscopy (AFM). No significant differences were observed between the surface area of cells cultured on gold substrates and Petri dishes, as indicated by measurements performed on SEM images. We also evaluated the electrophysiological compatibility of the cells through standard patch-clamp experiments by analyzing features such as the resting potential, membrane resistance, ionic currents, etc. Cells grown on both substrates showed no significant differences in their dependency on voltage, as well as in the magnitude of the Na+ and K+ current density; however, cells cultured on the gold substrate showed a lower membrane capacitance when compared to those grown on Petri dishes. By using two separate patch-clamp amplifiers, we were able to record the membrane current with the conventional patch-clamp technique and through the gold substrate simultaneously. Furthermore, the proposed technique allowed us to obtain simultaneous recordings of the electrical activity (such as action potentials firing) and the underlying membrane ionic currents. The excellent conductivity of gold makes it possible to overcome important difficulties found in conventional electrophysiological experiments such as those presented by the resistance of the electrolytic bath solution. We conclude that the technique here presented constitutes a solution to the problem of the simultaneous recording of electrical activity and the underlying ionic currents, which for decades, had been solved only partially.

In Vivo Measurement of Age-Related Stiffening in the Crystalline Lens by Brillouin Optical Microscopy

PubMed Central

Scarcelli, Giuliano; Kim, Pilhan; Yun, Seok Hyun

2011-01-01

Abtract The biophysical and biomechanical properties of the crystalline lens (e.g., viscoelasticity) have long been implicated in accommodation and vision problems, such as presbyopia and cataracts. However, it has been difficult to measure such parameters noninvasively. Here, we used in vivo Brillouin optical microscopy to characterize material acoustic properties at GHz frequency and measure the longitudinal elastic moduli of lenses. We obtained three-dimensional elasticity maps of the lenses in live mice, which showed biomechanical heterogeneity in the cortex and nucleus of the lens with high spatial resolution. An in vivo longitudinal study of mice over a period of 2 months revealed a marked age-related stiffening of the lens nucleus. We found remarkably good correlation (log-log linear) between the Brillouin elastic modulus and the Young's modulus measured by conventional mechanical techniques at low frequencies (∼1 Hz). Our results suggest that Brillouin microscopy is potentially useful for basic and animal research and clinical ophthalmology. PMID:21943436

In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.

PubMed

Scarcelli, Giuliano; Kim, Pilhan; Yun, Seok Hyun

2011-09-21

The biophysical and biomechanical properties of the crystalline lens (e.g., viscoelasticity) have long been implicated in accommodation and vision problems, such as presbyopia and cataracts. However, it has been difficult to measure such parameters noninvasively. Here, we used in vivo Brillouin optical microscopy to characterize material acoustic properties at GHz frequency and measure the longitudinal elastic moduli of lenses. We obtained three-dimensional elasticity maps of the lenses in live mice, which showed biomechanical heterogeneity in the cortex and nucleus of the lens with high spatial resolution. An in vivo longitudinal study of mice over a period of 2 months revealed a marked age-related stiffening of the lens nucleus. We found remarkably good correlation (log-log linear) between the Brillouin elastic modulus and the Young's modulus measured by conventional mechanical techniques at low frequencies (~1 Hz). Our results suggest that Brillouin microscopy is potentially useful for basic and animal research and clinical ophthalmology. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Inducible fluorescent speckle microscopy

PubMed Central

Aguiar, Paulo; Belsley, Michael; Maiato, Helder

2016-01-01

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

Inducible fluorescent speckle microscopy.

PubMed

Pereira, António J; Aguiar, Paulo; Belsley, Michael; Maiato, Helder

2016-01-18

The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. © 2016 Pereira et al.

Example-Based Super-Resolution Fluorescence Microscopy.

PubMed

Jia, Shu; Han, Boran; Kutz, J Nathan

2018-04-23

Capturing biological dynamics with high spatiotemporal resolution demands the advancement in imaging technologies. Super-resolution fluorescence microscopy offers spatial resolution surpassing the diffraction limit to resolve near-molecular-level details. While various strategies have been reported to improve the temporal resolution of super-resolution imaging, all super-resolution techniques are still fundamentally limited by the trade-off associated with the longer image acquisition time that is needed to achieve higher spatial information. Here, we demonstrated an example-based, computational method that aims to obtain super-resolution images using conventional imaging without increasing the imaging time. With a low-resolution image input, the method provides an estimate of its super-resolution image based on an example database that contains super- and low-resolution image pairs of biological structures of interest. The computational imaging of cellular microtubules agrees approximately with the experimental super-resolution STORM results. This new approach may offer potential improvements in temporal resolution for experimental super-resolution fluorescence microscopy and provide a new path for large-data aided biomedical imaging.

Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning of thick tissues

NASA Astrophysics Data System (ADS)

Cheng, Li-Chung; Chang, Chia-Yuan; Yen, Wei-Chung; Chen, Shean-Jen

2012-10-01

Conventional multiphoton microscopy employs beam scanning; however, in this study a microscope based on spatiotemporal focusing offering widefield multiphoton excitation has been developed to provide fast optical sectioning images. The microscope integrates a 10 kHz repetition rate ultrafast amplifier featuring strong instantaneous peak power (maximum 400 μJ/pulse at 90 fs pulse width) with a TE-cooled, ultra-sensitive photon detecting, electron multiplying charge-coupled device camera. This configuration can produce multiphoton excited images with an excitation area larger than 200 × 100 μm2 at a frame rate greater than 100 Hz. Brownian motions of fluorescent microbeads as small as 0.5 μm have been instantaneously observed with a lateral spatial resolution of less than 0.5 μm and an axial resolution of approximately 3.5 μm. Moreover, we combine the widefield multiphoton microscopy with structure illuminated technique named HiLo to reject the background scattering noise to get better quality for bioimaging.

Effect of combination ultrasonic and ball milling techniques of commercial fillers dispersion on mechanical properties of natural rubber (NR) latex films

NASA Astrophysics Data System (ADS)

Hamran, Noramirah; Rashid, Azura A.

2017-07-01

Commercial fillers such as silica and carbon black generally impart the reinforcing effects in dry rubber compound, but have an adverse effect on Natural rubber (NR) latex compounds. The addition of commercial fillers in NR latex has reduced the mechanical properties of NR latex films due to the destabilization effect in the NR latex compounds which govern by the dispersion quality, particle size and also the pH of the dispersion itself. The ball milling process is the conventional meth od of preparation of dispersions and ultrasonic has successfully used in preparation of nano fillers such as carbon nanotube (CNT). In this study the combination between the conventional methods; ball milling together the ultrasonic method were used to prepare the silica and carbon black dispersions. The different duration of ball milling (24, 48 and 72 hours) was compared with the ultrasonic method (30, 60, 90 and 120 minutes). The combination of ball milling and ultrasonic from the optimum individual technique was used to investigate the reduction of particle size of the fillers. The particle size analyzer, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) test were carried out to investigate the obtained particle size and the tensile and tear test were carried out to investigate the mechanical properties of the NR latex films. The reduction of filler particle size is expected to impart the properties of NR latex films.

Low-Temperature Growth and Doping of Mercury-Based II-Vi Multiple Quantum Well Structures by Molecular Beam Epitaxy

NASA Astrophysics Data System (ADS)

Lansari, Yamina

The growth of Hg-based single layers and multiple quantum well structures by conventional molecular beam epitaxy (MBE) and photoassisted MBE was studied. The use of photoassisted MBE, an epitaxial growth technique developed at NCSU, has resulted in a substantial reduction of the film growth temperature. Indeed, substrate temperatures 50 to 100^circC lower than those customarily used by others for conventional MBE growth of Hg-based layers were successfully employed. Photoassisted MBE allowed the preparation of excellent structural quality HgTe layers (FWHM for the (400) diffraction peak ~ 40 arcsec), HgCdTe layers (FWHM for the (400) diffraction peak ~ 14 arcsec), and HgTeCdTe superlattices (FWHM for the (400) diffraction peak ~ 28 arcsec). In addition, n-type and p-type modulation-doping of Hg-based multilayers was accomplished by photoassisted MBE. This technique has been shown to have a significant effect on the growth process kinetics as well as on the desorption rates of the film species, thereby affecting dopant incorporation mechanisms and allowing for the successful substitutional doping of the multilayer structures. Finally, surface morphology studies were completed using scanning electron microscopy (SEM) and Nomarsky optical microscopy to study the effects of substrate surface preparation, growth initiation, and growth parameters on the density of pyramidal hillocks, a common growth defect plaguing the Hg-based layers grown in the (100) direction. Conditions which minimize the hillock density for (100) film growth have been determined.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

Preparation and characterization of multi-layer biodegradable nanofibers by coaxial electrospinning and their potential for tissue engineering

NASA Astrophysics Data System (ADS)

Liu, Wenwen

As an evolution of conventional electrospinning, coaxial electrospinning became popular soon after its debut as a novel way to develop nanofibers with special structures, such as core-shell and hollow interior. In recent years, there has been an increasing interest in a modified coaxial electrospinning, tri-layer coaxial electrospinning, to develop more complex structures, such as multi-layer and nanowire-in-microtube. Previous studies have primarily concentrated on the fabrication of tri-layered inorganic fibers while studies on tri-layered coaxial polymeric fibers has not been reported until very recently. Our research focuses on the fabrication of core-shell and tri-layer structured biodegradable polymeric nanofibers with coaxial electrospinning. Different characterization methods have been applied to observe the internal structure in single nanofibers and the potential application of tri-layer coaxial electrospinning has been discussed. The material system consists of biodegradable natural polymer gelatin, synthetic polymers poly (epsilon-caprolactone) (PCL) and poly (lactic-co-glycolic acid) (PLGA). A uniquely designed three-needle concentric spinneret is developed to perform tri-layer coaxial electrospinning. Different kinds of core-shell structured nanofibers, including gelatin/PCL, PCL/gelatin, gelatin/PLGA and PCL/PLGA, have been fabricated with a customized coaxial electrospinning apparatus. Two kinds of tri-layer coaxial nanofibers, two-component ABA structured gelatin/PCL/gelatin biodegradable nanofibers and tri-component ABC structured gelatin/PCL/PLGA biodegradable nanofibers, have been developed with the customized three needle coaxial electrospinning setup. The core-shell and tri-layered structures of electrospun nanofibers have been characterized by several commonly used techniques, such as laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Besides the conventional methods, other newer techniques, including focused ion beam-scanning electron microscopy (FIB-SEM), super-resolution structured illumination microscopy (SR-SIM) and nanoscale-infrared spectroscopy (nano-IR), have been explored to investigate the internal structure in singles fibers. Additionally, the potential application of coaxial electrospinning in the fabrication of bioactive scaffolds for tissue engineering has been studied. Different kinds of coaxial nanofibers were fabricated and studied to determine the potential for BSA and growth factor release and some preliminary results were obtained.

Characterization of the Etna volcanic emissions through an active biomonitoring technique (moss-bags): part 2--morphological and mineralogical features.

PubMed

Calabrese, S; D'Alessandro, W

2015-01-01

Volcanic emissions were studied at Mount Etna (Italy) by using moss-bags technique. Mosses were exposed around the volcano at different distances from the active vents to evaluate the impact of volcanic emissions in the atmosphere. Morphology and mineralogy of volcanic particulate intercepted by mosses were investigated using scanning electron microscopy (SEM) equipped with energy dispersive spectrometer (EDS). Particles emitted during passive degassing activity from the two active vents, Bocca Nuova and North East Crater (BNC and NEC), were identified as silicates, sulfates and halide compounds. In addition to volcanic particles, we found evidences also of geogenic, anthropogenic and marine spray input. The study has shown the robustness of this active biomonitoring technique to collect particles, very useful in active volcanic areas characterized by continuous degassing and often not easily accessible to apply conventional sampling techniques. Copyright © 2014 Elsevier Ltd. All rights reserved.

The equation of state of 5-nitro-2,4-dihydro-1,2,4,-triazol-3-one determined via in-situ optical microscopy and interferometry measurements

DOE PAGES

Stavrou, Elissaios; Zaug, Joseph M.; Bastea, Sorin; ...

2016-04-07

Quasi-hydrostatic high-pressure equations of state (EOS) are typically determined, for crystalline solids, by measuring unit-cell volumes using x-ray diffraction (XRD) techniques. However, when characterizing low-symmetry materials with large unit cells, conventional XRD approaches may become problematic. To overcome this issue, we examined the utility of a "direct" approach toward determining high pressure material volume by measuring surface area and sample thickness using optical microscopy and interferometry (OMI) respectively. We have validated this experimental approach by comparing results obtained for TATB (2,4,6-triamino-1,3,5-trinitrobenzene) with an EOS determined from synchrotron XRD measurements; and, a good match is observed. We have measured the highmore » pressure EOS of 5-nitro-2,4-dihydro-1,2,4-triazol-3-one (α-NTO) up to 33 GPa. No high-pressure XRD EOS data have been published on α-NTO, probably due to its complex crystal structure. Furthermore, the results of this study suggest that OMI is a reliable and versatile alternative for determining EOSs, especially when conventional methodologies are impractical.« less

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

PubMed

Gopakumar, G; Hari Babu, K; Mishra, Deepak; Gorthi, Sai Siva; Sai Subrahmanyam, Gorthi R K

2017-01-01

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

The equation of state of 5-nitro-2,4-dihydro-1,2,4,-triazol-3-one determined via in-situ optical microscopy and interferometry measurements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stavrou, Elissaios, E-mail: stavrou1@llnl.gov; Zaug, Joseph M., E-mail: zaug1@llnl.gov; Bastea, Sorin

2016-04-07

Quasi-hydrostatic high-pressure equations of state (EOS) are typically determined, for crystalline solids, by measuring unit-cell volumes using x-ray diffraction (XRD) techniques. However, when characterizing low-symmetry materials with large unit cells, conventional XRD approaches may become problematic. To overcome this issue, we examined the utility of a “direct” approach toward determining high pressure material volume by measuring surface area and sample thickness using optical microscopy and interferometry (OMI), respectively. We have validated this experimental approach by comparing results obtained for 2,4,6-triamino-1,3,5-trinitrobenzene TATB with an EOS determined from synchrotron XRD measurements; and, a good match is observed. We have measured the high pressure EOS of 5-nitro-2,4-dihydro-1,2,4,-triazol-3-one (α-NTO) upmore » to 28 GPa. No high-pressure XRD EOS data have been published on α-NTO, probably due to its complex crystal structure. The results of this study suggest that OMI is a reliable and versatile alternative for determining EOSs, especially when conventional methodologies are impractical.« less

Superresolution imaging of Drosophila tissues using expansion microscopy.

PubMed

Jiang, Nan; Kim, Hyeon-Jin; Chozinski, Tyler J; Azpurua, Jorge E; Eaton, Benjamin A; Vaughan, Joshua C; Parrish, Jay Z

2018-06-15

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.

Silicifying Biofilm Exopolymers on a Hot-Spring Microstromatolite: Templating Nanometer-Thick Laminae

NASA Astrophysics Data System (ADS)

Handley, Kim M.; Turner, Sue J.; Campbell, Kathleen A.; Mountain, Bruce W.

2008-08-01

Exopolymeric substances (EPS) are an integral component of microbial biofilms; however, few studies have addressed their silicification and preservation in hot-spring deposits. Through comparative analyses with the use of a range of microscopy techniques, we identified abundant EPS significant to the textural development of spicular, microstromatolitic, siliceous sinter at Champagne Pool, Waiotapu, New Zealand. Examination of biofilms coating sinter surfaces by confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM), cryo-scanning electron microscopy (cryo-SEM), and transmission electron microscopy (TEM) revealed contraction of the gelatinous EPS matrix into films (approximately 10 nm thick) or fibrillar structures, which is common in conventional SEM analyses and analogous to products of naturally occurring desiccation. Silicification of fibrillar EPS contributed to the formation of filamentous sinter. Matrix surfaces or dehydrated films templated sinter laminae (nanometers to microns thick) that, in places, preserved fenestral voids beneath. Laminae of similar thickness are, in general, common to spicular geyserites. This is the first report to demonstrate EPS templation of siliceous stromatolite laminae. Considering the ubiquity of biofilms on surfaces in hot-spring environments, EPS silicification studies are likely to be important to a better understanding of the origins of laminae in other modern and ancient stromatolitic sinters, and EPS potentially may serve as biosignatures in extraterrestrial rocks.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications

NASA Astrophysics Data System (ADS)

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W.; Dokmeci, Mehmet Remzi; Boyden, Edward S.; Khademhosseini, Ali

2016-03-01

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such “hybrid microscopy” methods—combining physical and optical magnifications—can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes (“mini-microscopes”), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics—a process we refer to as Expansion Mini-Microscopy (ExMM)—is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Detection, imaging, and kinetics of sub-micron organelles of chondrocytes by multiple beam interference microscopy

NASA Astrophysics Data System (ADS)

Joshi, Narahari V.; Medina, Honorio; Barboza, J. M.; Colantuoni, Gladys; Quintero, Maritza

2004-07-01

Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.

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

NASA Astrophysics Data System (ADS)

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

2016-03-01

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

Interference techniques in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dogan, Mehmet

We developed a set of interference-based optical microscopy techniques to study biological structures through nanometer-scale axial localization of fluorescent biomarkers. Spectral self-interference fluorescence microscopy (SSFM) utilizes interference of direct and reflected waves emitted from fluorescent molecules in the vicinity of planar reflectors to reveal the axial position of the molecules. A comprehensive calculation algorithm based on Green's function formalism is presented to verify the validity of approximations used in a far-field approach that describes the emission of fluorescent markers near interfaces. Using the validated model, theoretical limits of axial localization were determined with emphasis given to numerical aperture (NA) dependence of localization uncertainty. SSFM was experimentally demonstrated in conformational analysis of nucleoproteins. In particular, interaction between surface-tethered 75-mer double strand DNA and integration host factor (IHF) protein was probed on Si-SiO2 substrates by determining the axial position of fluorescent labels attached to the free ends of DNA molecules. Despite its sub-nanometer precision axial localization capability, SSFM lacks high lateral resolution due to the low-NA requirement for planar reflectors. We developed a second technique, 4Pi-SSFM, which improves the lateral resolution of a conventional SSFM system by an order of magnitude while achieving nanometer-scale axial localization precision. Using two opposing high-NA objectives, fluorescence signal is interferometrically collected and spectral interference pattern is recorded. Axial position of emitters is found from analysis of the spectra. The 4Pi-SSFM technique was experimentally demonstrated by determining the surface profiles of fabricated glass surfaces and outer membranes of Shigella, a type of Gram-negative bacteria. A further discussion is presented to localize surface O antigen, which is an important oligosaccharide structure in the virulence mechanism of the Gram-negative bacteria, including E. coli and Shigella.

Structural, optical and AFM characterization of PVA:La3+ polymer films

NASA Astrophysics Data System (ADS)

Ali, F. M.; Maiz, F.

2018-02-01

In this paper the structural and optical properties of pure Polyvinyl alcohol (PVA) and La3+-doped PVA films in the concentration range of 4%, 12% and 20% weight percent of Lanthanum were prepared by the conventional casting technique. X-ray diffraction pattern and atomic force microscopy studies of the investigated samples reveal their semi-crystalline nature. It is found that, absorption coefficient and cluster size of lanthanum:PVA composite increase with increasing salt concentration. However, the optical energy gap shows a slight decreasing trend.

A review of advantages of high-efficiency X-ray spectrum imaging for analysis of nanostructured ferritic alloys

DOE PAGES

Parish, Chad M.; Miller, Michael K.

2014-12-09

Nanostructured ferritic alloys (NFAs) exhibit complex microstructures consisting of 100-500 nm ferrite grains, grain boundary solute enrichment, and multiple populations of precipitates and nanoclusters (NCs). Understanding these materials' excellent creep and radiation-tolerance properties requires a combination of multiple atomic-scale experimental techniques. Recent advances in scanning transmission electron microscopy (STEM) hardware and data analysis methods have the potential to revolutionize nanometer to micrometer scale materials analysis. The application of these methods is applied to NFAs as a test case and is compared to both conventional STEM methods as well as complementary methods such as scanning electron microscopy and atom probe tomography.more » In this paper, we review past results and present new results illustrating the effectiveness of latest-generation STEM instrumentation and data analysis.« less

Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

PubMed

Starborg, Tobias; Kadler, Karl E

2015-03-01

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development. © 2015 Wiley Periodicals, Inc.

Movies of cellular and sub-cellular motion by digital holographic microscopy.

PubMed

Mann, Christopher J; Yu, Lingfeng; Kim, Myung K

2006-03-23

Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration. Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

Whole-animal imaging with high spatio-temporal resolution

NASA Astrophysics Data System (ADS)

Chhetri, Raghav; Amat, Fernando; Wan, Yinan; Höckendorf, Burkhard; Lemon, William C.; Keller, Philipp J.

2016-03-01

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of ≤ 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Two-color SERS microscopy for protein co-localization in prostate tissue with primary antibody-protein A/G-gold nanocluster conjugates

NASA Astrophysics Data System (ADS)

Salehi, Mohammad; Schneider, Lilli; Ströbel, Philipp; Marx, Alexander; Packeisen, Jens; Schlücker, Sebastian

2014-01-01

SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates.SERS microscopy is a novel staining technique in immunohistochemistry, which is based on antibodies labeled with functionalized noble metal colloids called SERS labels or nanotags for optical detection. Conventional covalent bioconjugation of these SERS labels cannot prevent blocking of the antigen recognition sites of the antibody. We present a rational chemical design for SERS label-antibody conjugates which addresses this issue. Highly sensitive, silica-coated gold nanoparticle clusters as SERS labels are non-covalently conjugated to primary antibodies by using the chimeric protein A/G, which selectively recognizes the Fc part of antibodies and therefore prevents blocking of the antigen recognition sites. In proof-of-concept two-color imaging experiments for the co-localization of p63 and PSA on non-neoplastic prostate tissue FFPE specimens, we demonstrate the specificity and signal brightness of these rationally designed primary antibody-protein A/G-gold nanocluster conjugates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05890e

Imaging live cells at high spatiotemporal resolution for lab-on-a-chip applications.

PubMed

Chin, Lip Ket; Lee, Chau-Hwang; Chen, Bi-Chang

2016-05-24

Conventional optical imaging techniques are limited by the diffraction limit and difficult-to-image biomolecular and sub-cellular processes in living specimens. Novel optical imaging techniques are constantly evolving with the desire to innovate an imaging tool that is capable of seeing sub-cellular processes in a biological system, especially in three dimensions (3D) over time, i.e. 4D imaging. For fluorescence imaging on live cells, the trade-offs among imaging depth, spatial resolution, temporal resolution and photo-damage are constrained based on the limited photons of the emitters. The fundamental solution to solve this dilemma is to enlarge the photon bank such as the development of photostable and bright fluorophores, leading to the innovation in optical imaging techniques such as super-resolution microscopy and light sheet microscopy. With the synergy of microfluidic technology that is capable of manipulating biological cells and controlling their microenvironments to mimic in vivo physiological environments, studies of sub-cellular processes in various biological systems can be simplified and investigated systematically. In this review, we provide an overview of current state-of-the-art super-resolution and 3D live cell imaging techniques and their lab-on-a-chip applications, and finally discuss future research trends in new and breakthrough research areas of live specimen 4D imaging in controlled 3D microenvironments.

Compact Microscope Imaging System With Intelligent Controls Improved

NASA Technical Reports Server (NTRS)

McDowell, Mark

2004-01-01

The Compact Microscope Imaging System (CMIS) with intelligent controls is a diagnostic microscope analysis tool with intelligent controls for use in space, industrial, medical, and security applications. This compact miniature microscope, which can perform tasks usually reserved for conventional microscopes, has unique advantages in the fields of microscopy, biomedical research, inline process inspection, and space science. Its unique approach integrates a machine vision technique with an instrumentation and control technique that provides intelligence via the use of adaptive neural networks. The CMIS system was developed at the NASA Glenn Research Center specifically for interface detection used for colloid hard spheres experiments; biological cell detection for patch clamping, cell movement, and tracking; and detection of anode and cathode defects for laboratory samples using microscope technology.

Imaging of the human tooth cementum ultrastructure of archeological teeth, using hard x-ray microtomography to determine age-at-death and stress periods

NASA Astrophysics Data System (ADS)

Mani-Caplazi, Gabriela; Schulz, Georg; Deyhle, Hans; Hotz, Gerhard; Vach, Werner; Wittwer-Backofen, Ursula; Müller, Bert

2017-09-01

Tooth cementum annulation (TCA) is used by anthropologists to decipher age-at-death and stress periods based on yearly deposited incremental lines (ILs). The destructive aspect of the TCA method, which requires cutting the tooth root in sections to display the ILs, using transmission light microscopy, can be problematic for archeological teeth, and so a non-invasive imaging technique is preferred. The purpose of this study is to evaluate conventional micro computed tomography (μCT) and synchrotron radiation-based X-ray micro computed tomography (SRμCT) as a non-destructive technique to explore the tooth cementum ultrastructure and to display ILs. Seven archeological teeth from the Basel- Spitalfriedhof collection (patients died between 1845 and 1868 in the city hospital) were selected for the μCT experiments. This collection is considered a unique worldwide reference series in the anthropological science community, due to the high level of documented life history data in the medical files and the additionally collected and verified birth history by genealogists. The results demonstrate that the conventional μCT is complementary to the SRμCT allowing to prescreen the teeth using conventional μCT to identify the appropriate specimens and areas for the SRμCT measurements. SRμCT displayed cementum ring structure corresponding to the ILs in the microscope view in archeological teeth in a non-invasive fashion with the potential for more accurate assessments of ILs compared to conventional techniques. The ILs were mainly clearly visible, and it was possible to count them for age-at-death assessment and identify qualitatively irregular ILs which could constitute stress markers.

The evolution of structured illumination microscopy in studies of HIV.

PubMed

Marno, Kelly; Al'Zoubi, Lara; Pearson, Matthew; Posch, Markus; McKnight, Áine; Wheeler, Ann P

2015-10-15

The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years. Copyright © 2015. Published by Elsevier Inc.

Corneal confocal microscopy following conventional, transepithelial, and accelerated corneal collagen cross-linking procedures for keratoconus.

PubMed

Touboul, David; Efron, Nathan; Smadja, David; Praud, Delphine; Malet, Florence; Colin, Joseph

2012-11-01

To compare early corneal healing following conventional, transepithelial, and accelerated corneal collagen cross-linking (CXL) protocols. Twenty-four patients with progressive keratoconus were divided into three groups to receive conventional, transepithelial, or accelerated CXL. In vivo corneal confocal microscopy was performed on each patient preoperatively and at 1, 3, and 6 months postoperatively. Closure of the epithelial wound was complete 3 days following conventional and accelerated CXL. The subbasal nerve plexus was essentially obliterated immediately following conventional and accelerated CXL, and virtually no nerve fibers had regenerated by 6 months. The anterior stroma showed significant changes 1 month following conventional CXL; these changes were similar but more pronounced following accelerated CXL. Observed stromal changes included complete obliteration of keratocytes, increased tissue reflectivity, a honeycomb-like appearance, and circular lacunae. Some recovery of keratocyte density was noted after 6 months. These changes were less pronounced in the mid-stroma, and there were no apparent changes to the posterior stroma or endothelium. The cornea appeared to be unaltered following transepithelial CXL. In vivo corneal confocal microscopy analysis of the postoperative impact of CXL on the cornea revealed clear differences among conventional, accelerated, and transepithelial CXL protocols. Accelerated CXL had a greater impact than conventional CXL on the anterior cornea, whereas transepithelial CXL did not appear to alter corneal morphology. Copyright 2012, SLACK Incorporated.

Laser microfabrication of biomedical devices: time-resolved microscopy of the printing process

NASA Astrophysics Data System (ADS)

Serra, P.; Patrascioiu, A.; Fernández-Pradas, J. M.; Morenza, J. L.

2013-04-01

Laser printing constitutes an interesting alternative to more conventional printing techniques in the microfabrication of biomedical devices. The principle of operation of most laser printing techniques relies on the highly localized absorption of strongly focused laser pulses in the close proximity of the free surface of the liquid to be printed. This leads to the generation of a cavitation bubble which further expansion results in the ejection of a small fraction of the liquid, giving place to the deposition of a well-defined droplet onto a collector substrate. Laser printing has proved feasible for printing biological materials, from single-stranded DNA to proteins, and even living cells and microorganisms, with high degrees of resolution and reproducibility. In consequence, laser printing appears to be an excellent candidate for the fabrication of biological microdevices, such as DNA and protein microarrays, or miniaturized biosensors. The optimization of the performances of laser printing techniques requires a detailed knowledge of the dynamics of liquid transfer. Time-resolved microscopy techniques play a crucial role in this concern, since they allow tracking the evolution of the ejected material with excellent time and spatial resolution. Investigations carried out up to date have shown that liquid ejection proceeds through the formation of long, thin and stable liquid jets. In this work the different approaches used so far for monitoring liquid ejection during laser printing are considered, and it is shown how these techniques make possible to understand the complex dynamics involved in the process.

Quantitative X-ray Differential Interference Contrast Microscopy

NASA Astrophysics Data System (ADS)

Nakamura, Takashi

Full-field soft x-ray microscopes are widely used in many fields of sciences. Advances in nanofabrication technology enabled short wavelength focusing elements with significantly improved spatial resolution. In the soft x-ray spectral region, samples as small as 12 nm can be resolved using micro zone-plates as the objective lens. In addition to conventional x-ray microscopy in which x-ray absorption difference provides the image contrast, phase contrast mechanisms such as differential phase contrast (DIC) and Zernike phase contrast have also been demonstrated These phase contrast imaging mechanisms are especially attractive at the x-ray wavelengths where phase contrast of most materials is typically 10 times stronger than the absorption contrast. With recent progresses in plasma-based x- ray sources and increasing accessibility to synchrotron user facilities, x-ray microscopes are quickly becoming standard measurement equipment in the laboratory. To further the usefulness of x-ray DIC microscopy this thesis explicitly addresses three known issues with this imaging modality by introducing new techniques and devices First, as opposed to its visible-light counterpart, no quantitative phase imaging technique exists for x-ray DIC microscopy. To address this issue, two nanoscale x-ray quantitative phase imaging techniques, using exclusive OR (XOR) patterns and zone-plate doublets, respectively, are proposed. Unlike existing x-ray quantitative phase imaging techniques such as Talbot interferometry and ptychography, no dedicated experimental setups or stringent illumination coherence are needed for quantitative phase retrieval. Second, to the best of our knowledge, no quantitative performance characterization of DIC microscopy exists to date. Therefore the imaging system's response to sample's spatial frequency is not known In order to gain in-depth understanding of this imaging modality, performance of x-ray DIC microscopy is quantified using modulation transfer function. A new illumination apparatus required for the transfer function analysis under partially coherent illumination is also proposed. Such a characterization is essential for a proper selection of DIC optics for various transparent samples under study. Finally, optical elements used for x-ray DIC microscopy are highly absorptive and high brilliance x-ray sources such as synchrotrons are generally needed for image contrast. To extend the use of x-ray DIC microscopy to a wider variety of applications, a high efficiency large numerical aperture optical element consisting of high reflective Bragg reflectors is proposed. Using Bragg reflectors, which have 70% ˜99% reflectivity at extreme ultraviolet and soft x-rays for all angles of glancing incidence, the first order focusing efficiency is expected to increase by ˜ 8 times compared to that of a typical Fresnel zone-plate. This thesis contributes to current nanoscale x-ray phase contrast imaging research and provides new insights for biological, material, and magnetic sciences

Spectral interferometry for morphological imaging in in vitro fertilization (IVF) (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhu, Yizheng; Li, Chengshuai

2016-03-01

Morphological assessment of spermatozoa is of critical importance for in vitro fertilization (IVF), especially intracytoplasmic sperm injection (ICSI)-based IVF. In ICSI, a single sperm cell is selected and injected into an egg to achieve fertilization. The quality of the sperm cell is found to be highly correlated to IVF success. Sperm morphology, such as shape, head birefringence and motility, among others, are typically evaluated under a microscope. Current observation relies on conventional techniques such as differential interference contrast microscopy and polarized light microscopy. Their qualitative nature, however, limits the ability to provide accurate quantitative analysis. Here, we demonstrate quantitative morphological measurement of sperm cells using two types of spectral interferometric techniques, namely spectral modulation interferometry and spectral multiplexing interferometry. Both are based on spectral-domain low coherence interferometry, which is known for its exquisite phase determination ability. While spectral modulation interferometry encodes sample phase in a single spectrum, spectral multiplexing interferometry does so for sample birefringence. Therefore they are capable of highly sensitive phase and birefringence imaging. These features suit well in the imaging of live sperm cells, which are small, dynamic objects with only low to moderate levels of phase and birefringence contrast. We will introduce the operation of both techniques and demonstrate their application to measuring the phase and birefringence morphology of sperm cells.

Application of microscopy technique and high-performance liquid chromatography for quality assessment of the flower bud of Tussilago farfara L. (Kuandonghua)

PubMed Central

Li, Da; Liang, Li; Zhang, Jing; Kang, Tingguo

2015-01-01

Background: Quality control is one of the bottleneck problems limiting the application and development of traditional Chinese medicine (TCM). In recent years, microscopy and high-performance liquid chromatography (HPLC) techniques have been frequently applied in the quality control of TCM. However, studies combining conventional microscopy and HPLC techniques for the quality control of the flower bud of Tussilago farfara L. (Kuandonghua) have not been reported. Objective: This study was undertaken to evaluate the quality of the flower bud of T. farfara L. and to establish the relationships between the quantity of pollen grains and four main bioactive constituents: tussilagone, chlorogenic acid, rutin and isoquercitrin. Materials and Methods: In this study, microscopic examination was used to quantify microscopic characteristics of the flower bud of T. farfara L., and the chemical components were determined by HPLC. The data were analyzed by Statistical Package for the Social Sciences statistics software. Results: The results of the analysis showed that tussilagone, chlorogenic acid, rutin and isoquercitrin were significantly correlated with the quantity of pollen grains in the flower bud of T. farfara L. There is a positive correlation between them. From these results, it can be deduced that the flower bud of T. farfara L. with a greater quantity of pollen grains should be of better quality. Conclusion: The study showed that the established method can be helpful for evaluating the quality of the flower bud of T. farfara L. based on microscopic characteristic constants and chemical quantitation. PMID:26246737

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Infrared Microspectroscopy: A Multiple-Screening Platform for Investigating Single-Cell Biochemical Perturbations upon Prion Infection

PubMed Central

2011-01-01

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrPSc) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrPC, into nascent PrPSc. The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level. PMID:22778865

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy.

PubMed

Yang, Soo In; George, Graham N; Lawrence, John R; Kaminskyj, Susan G W; Dynes, James J; Lai, Barry; Pickering, Ingrid J

2016-10-04

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to the same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se 0 ). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se 0 using the Se L III edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50-700 nm, suggesting Se 0 nanoparticles. The intimate association of Se 0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X-ray Fluorescence Imaging and Scanning Transmission X-ray Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Soo In; George, Graham N.; Lawrence, John R.

2016-10-04

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to themore » same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se0). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se0 using the Se LIII edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50–700 nm, suggesting Se0 nanoparticles. The intimate association of Se0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.« less

Infrared microspectroscopy: a multiple-screening platform for investigating single-cell biochemical perturbations upon prion infection.

PubMed

Didonna, Alessandro; Vaccari, Lisa; Bek, Alpan; Legname, Giuseppe

2011-03-16

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of prions in the central nervous system. The pathogenic prion (PrP(Sc)) possesses the capability to convert the host-encoded cellular isoform of the prion protein, PrP(C), into nascent PrP(Sc). The present work aims at providing novel insight into cellular response upon prion infection evidenced by synchrotron radiation infrared microspectroscopy (SR-IRMS). This non-invasive, label-free analytical technique was employed to investigate the biochemical perturbations undergone by prion infected mouse hypothalamic GT1-1 cells at the cellular and subcellular level. A decrement in total cellular protein content upon prion infection was identified by infrared (IR) whole-cell spectra and validated by bicinchoninic acid assay and single-cell volume analysis by atomic force microscopy (AFM). Hierarchical cluster analysis (HCA) of IR data discriminated between infected and uninfected cells and allowed to deduce an increment of lysosomal bodies within the cytoplasm of infected GT1-1 cells, a hypothesis further confirmed by SR-IRMS at subcellular spatial resolution and fluorescent microscopy. The purpose of this work, therefore, consists of proposing IRMS as a powerful multiscreening platform, drawing on the synergy with conventional biological assays and microscopy techniques in order to increase the accuracy of investigations performed at the single-cell level.

Adherence of Candida to complete denture surfaces in vitro: A comparison of conventional and CAD/CAM complete dentures

PubMed Central

Al-mejrad, Lamya A.; Albarrag, Ahmed M.

2017-01-01

PURPOSE The goal of this study was to compare the adhesion of Candida albicans to the surfaces of CAD/CAM and conventionally fabricated complete denture bases. MATERIALS AND METHODS Twenty discs of acrylic resin poly (methyl methacrylate) were fabricated with CAD/CAM and conventional procedures (heat-polymerized acrylic resin). The specimens were divided into two groups: 10 discs were fabricated using the CAD/CAM procedure (Wieland Digital Denture Ivoclar Vivadent), and 10 discs were fabricated using a conventional flasking and pressure-pack technique. Candida colonization was performed on all the specimens using four Candida albicans isolates. The difference in Candida albicans adhesion on the discs was evaluated. The number of adherent yeast cells was calculated by the colony-forming units (CFU) and by Fluorescence microscopy. RESULTS There was a significant difference in the adhesion of Candida albicans to the complete denture bases created with CAD/CAM and the adhesion to those created with the conventional procedure. The CAD/CAM denture bases exhibited less adhesion of Candida albicans than did the denture bases created with the conventional procedure (P<.05). CONCLUSION The CAD/CAM procedure for fabricating complete dentures showed promising potential for reducing the adherence of Candida to the denture base surface. Clinical Implications. Complete dentures made with the CAD/CAM procedure might decrease the incidence of denture stomatitis compared with conventional dentures. PMID:29142649

Adherence of Candida to complete denture surfaces in vitro: A comparison of conventional and CAD/CAM complete dentures.

PubMed

Al-Fouzan, Afnan F; Al-Mejrad, Lamya A; Albarrag, Ahmed M

2017-10-01

The goal of this study was to compare the adhesion of Candida albicans to the surfaces of CAD/CAM and conventionally fabricated complete denture bases. Twenty discs of acrylic resin poly (methyl methacrylate) were fabricated with CAD/CAM and conventional procedures (heat-polymerized acrylic resin). The specimens were divided into two groups: 10 discs were fabricated using the CAD/CAM procedure (Wieland Digital Denture Ivoclar Vivadent), and 10 discs were fabricated using a conventional flasking and pressure-pack technique. Candida colonization was performed on all the specimens using four Candida albicans isolates. The difference in Candida albicans adhesion on the discs was evaluated. The number of adherent yeast cells was calculated by the colony-forming units (CFU) and by Fluorescence microscopy. There was a significant difference in the adhesion of Candida albicans to the complete denture bases created with CAD/CAM and the adhesion to those created with the conventional procedure. The CAD/CAM denture bases exhibited less adhesion of Candida albicans than did the denture bases created with the conventional procedure ( P <.05). The CAD/CAM procedure for fabricating complete dentures showed promising potential for reducing the adherence of Candida to the denture base surface. Clinical Implications. Complete dentures made with the CAD/CAM procedure might decrease the incidence of denture stomatitis compared with conventional dentures.

Mobile microscopy as a screening tool for oral cancer in India: A pilot study.

PubMed

Skandarajah, Arunan; Sunny, Sumsum P; Gurpur, Praveen; Reber, Clay D; D'Ambrosio, Michael V; Raghavan, Nisheena; James, Bonney Lee; Ramanjinappa, Ravindra D; Suresh, Amritha; Kandasarma, Uma; Birur, Praveen; Kumar, Vinay V; Galmeanu, Honorius-Cezar; Itu, Alexandru Mihail; Modiga-Arsu, Mihai; Rausch, Saskia; Sramek, Maria; Kollegal, Manohar; Paladini, Gianluca; Kuriakose, Moni; Ladic, Lance; Koch, Felix; Fletcher, Daniel

2017-01-01

Oral cancer is the most common type of cancer among men in India and other countries in South Asia. Late diagnosis contributes significantly to this mortality, highlighting the need for effective and specific point-of-care diagnostic tools. The same regions with high prevalence of oral cancer have seen extensive growth in mobile phone infrastructure, which enables widespread access to telemedicine services. In this work, we describe the evaluation of an automated tablet-based mobile microscope as an adjunct for telemedicine-based oral cancer screening in India. Brush biopsy, a minimally invasive sampling technique was combined with a simplified staining protocol and a tablet-based mobile microscope to facilitate local collection of digital images and remote evaluation of the images by clinicians. The tablet-based mobile microscope (CellScope device) combines an iPad Mini with collection optics, LED illumination and Bluetooth-controlled motors to scan a slide specimen and capture high-resolution images of stained brush biopsy samples. Researchers at the Mazumdar Shaw Medical Foundation (MSMF) in Bangalore, India used the instrument to collect and send randomly selected images of each slide for telepathology review. Evaluation of the concordance between gold standard histology, conventional microscopy cytology, and remote pathologist review of the images was performed as part of a pilot study of mobile microscopy as a screening tool for oral cancer. Results indicated that the instrument successfully collected images of sufficient quality to enable remote diagnoses that show concordance with existing techniques. Further studies will evaluate the effectiveness of oral cancer screening with mobile microscopy by minimally trained technicians in low-resource settings.

Comparison of microscopy, ELISA, and real-time PCR for detection of Giardia intestinalis in human stool specimens

PubMed

Beyhan, Yunus Emre; Taş Cengiz, Zeynep

2017-08-23

Background/aim: This study included patients who had digestive system complaints between August 2015 and October 2015. The research was designed to compare conventional microscopy with an antigen detection ELISA kit and the TaqMan-based real-time PCR (RT-PCR) technique for detection of Giardia intestinalis in human stool specimens. Materials and methods: Samples were concentrated by formalin-ether sedimentation technique and microscopic examinations were carried out on wet mount slides. A commercially available ELISA kit (Giardia CELISA, Cellabs, Brookvale, Australia) was used for immunoassay. DNA was extracted from fecal samples of about 200 mg using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and the LightCycler Nano system (Roche Diagnostics, Mannheim, Germany) was used for the TaqMan-based RT-PCR assay. Results: A total of 94 stool samples, 38 of them diagnosed positive (40.4%) and 56 of them diagnosed negative by microscopy, were selected for evaluation by antigen detection and molecular assays. The prevalence of G. intestinalis infection was found as 46.8% (n: 44) and 79.8% (n: 75) by ELISA and RT-PCR, respectively. RT-PCR revealed by far the highest positivity rate compared to the other two methods. The difference between these methods was found to be statistically significant (P < 0.05). In comparison to PCR, the sensitivity and specificity of microscopy and ELISA were 50.7% and 100% and 53.3% and 79%, respectively. Conclusion: RT-PCR seems to be much more sensitive and beneficial for rapid and accurate diagnosis of G. intestinalis in human stools.

Review of advanced imaging techniques

PubMed Central

Chen, Yu; Liang, Chia-Pin; Liu, Yang; Fischer, Andrew H.; Parwani, Anil V.; Pantanowitz, Liron

2012-01-01

Pathology informatics encompasses digital imaging and related applications. Several specialized microscopy techniques have emerged which permit the acquisition of digital images (“optical biopsies”) at high resolution. Coupled with fiber-optic and micro-optic components, some of these imaging techniques (e.g., optical coherence tomography) are now integrated with a wide range of imaging devices such as endoscopes, laparoscopes, catheters, and needles that enable imaging inside the body. These advanced imaging modalities have exciting diagnostic potential and introduce new opportunities in pathology. Therefore, it is important that pathology informaticists understand these advanced imaging techniques and the impact they have on pathology. This paper reviews several recently developed microscopic techniques, including diffraction-limited methods (e.g., confocal microscopy, 2-photon microscopy, 4Pi microscopy, and spatially modulated illumination microscopy) and subdiffraction techniques (e.g., photoactivated localization microscopy, stochastic optical reconstruction microscopy, and stimulated emission depletion microscopy). This article serves as a primer for pathology informaticists, highlighting the fundamentals and applications of advanced optical imaging techniques. PMID:22754737

STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

PubMed

Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue

PubMed Central

Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B. Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories. PMID:25025184

Surface-specific additive manufacturing test artefacts

NASA Astrophysics Data System (ADS)

Townsend, Andrew; Racasan, Radu; Blunt, Liam

2018-06-01

Many test artefact designs have been proposed for use with additive manufacturing (AM) systems. These test artefacts have primarily been designed for the evaluation of AM form and dimensional performance. A series of surface-specific measurement test artefacts designed for use in the verification of AM manufacturing processes are proposed here. Surface-specific test artefacts can be made more compact because they do not require the large dimensions needed for accurate dimensional and form measurements. The series of three test artefacts are designed to provide comprehensive information pertaining to the manufactured surface. Measurement possibilities include deviation analysis, surface texture parameter data generation, sub-surface analysis, layer step analysis and build resolution comparison. The test artefacts are designed to provide easy access for measurement using conventional surface measurement techniques, for example, focus variation microscopy, stylus profilometry, confocal microscopy and scanning electron microscopy. Additionally, the test artefacts may be simply visually inspected as a comparative tool, giving a fast indication of process variation between builds. The three test artefacts are small enough to be included in every build and include built-in manufacturing traceability information, making them a convenient physical record of the build.

Nanoscale nuclear architecture for cancer diagnosis beyond pathology via spatial-domain low-coherence quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Uttam, Shikhar; Staton, Kevin; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

2010-11-01

Definitive diagnosis of malignancy is often challenging due to limited availability of human cell or tissue samples and morphological similarity with certain benign conditions. Our recently developed novel technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM)-overcomes the technical difficulties and enables us to obtain quantitative information about cell nuclear architectural characteristics with nanoscale sensitivity. We explore its ability to improve the identification of malignancy, especially in cytopathologically non-cancerous-appearing cells. We perform proof-of-concept experiments with an animal model of colorectal carcinogenesis-APCMin mouse model and human cytology specimens of colorectal cancer. We show the ability of in situ nanoscale nuclear architectural characteristics in identifying cancerous cells, especially in those labeled as ``indeterminate or normal'' by expert cytopathologists. Our approach is based on the quantitative analysis of the cell nucleus on the original cytology slides without additional processing, which can be readily applied in a conventional clinical setting. Our simple and practical optical microscopy technique may lead to the development of novel methods for early detection of cancer.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

PubMed

Huang, Shixin; Makarem, Mohamadamin; Kiemle, Sarah N; Hamedi, Hossein; Sau, Moujhuri; Cosgrove, Daniel J; Kim, Seong H

2018-05-17

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

The application of Fresnel zone plate based projection in optofluidic microscopy.

PubMed

Wu, Jigang; Cui, Xiquan; Lee, Lap Man; Yang, Changhuei

2008-09-29

Optofluidic microscopy (OFM) is a novel technique for low-cost, high-resolution on-chip microscopy imaging. In this paper we report the use of the Fresnel zone plate (FZP) based projection in OFM as a cost-effective and compact means for projecting the transmission through an OFM's aperture array onto a sensor grid. We demonstrate this approach by employing a FZP (diameter = 255 microm, focal length = 800 microm) that has been patterned onto a glass slide to project the transmission from an array of apertures (diameter = 1 microm, separation = 10 microm) onto a CMOS sensor. We are able to resolve the contributions from 44 apertures on the sensor under the illumination from a HeNe laser (wavelength = 633 nm). The imaging quality of the FZP determines the effective field-of-view (related to the number of resolvable transmissions from apertures) but not the image resolution of such an OFM system--a key distinction from conventional microscope systems. We demonstrate the capability of the integrated system by flowing the protist Euglena gracilis across the aperture array microfluidically and performing OFM imaging of the samples.

High-definition X-ray fluorescence elemental mapping of paintings.

PubMed

Howard, Daryl L; de Jonge, Martin D; Lau, Deborah; Hay, David; Varcoe-Cocks, Michael; Ryan, Chris G; Kirkham, Robin; Moorhead, Gareth; Paterson, David; Thurrowgood, David

2012-04-03

A historical self-portrait painted by Sir Arthur Streeton (1867-1943) has been studied with fast-scanning X-ray fluorescence microscopy using synchrotron radiation. One of the technique's unique strengths is the ability to reveal metal distributions in the pigments of underlying brushstrokes, thus providing information critical to the interpretation of a painting. We have applied the nondestructive technique with the event-mode Maia X-ray detector, which has the capability to record elemental maps at megapixels per hour with the full X-ray fluorescence spectrum collected per pixel. The painting poses a difficult challenge to conventional X-ray analysis, because it was completely obscured with heavy brushstrokes of highly X-ray absorptive lead white paint (2PbCO(3)·Pb(OH)(2)) by the artist, making it an excellent candidate for the application of the synchrotron-based technique. The 25 megapixel elemental maps were successfully observed through the lead white paint across the 200 × 300 mm(2) scan area. The sweeping brushstrokes of the lead white overpaint contributed significant detrimental structure to the elemental maps. A corrective procedure was devised to enhance the visualization of the elemental maps by using the elastic X-ray scatter as a proxy for the lead white overpaint. We foresee the technique applied to the most demanding of culturally significant artworks where conventional analytical methods are inadequate.

Novel Techniques for Pulsed Field Gradient NMR Measurements

NASA Astrophysics Data System (ADS)

Brey, William Wallace

Pulsed field gradient (PFG) techniques now find application in multiple quantum filtering and diffusion experiments as well as in magnetic resonance imaging and spatially selective spectroscopy. Conventionally, the gradient fields are produced by azimuthal and longitudinal currents on the surfaces of one or two cylinders. Using a series of planar units consisting of azimuthal and radial current elements spaced along the longitudinal axis, we have designed gradient coils having linear regions that extend axially nearly to the ends of the coil and to more than 80% of the inner radius. These designs locate the current return paths on a concentric cylinder, so the coils are called Concentric Return Path (CRP) coils. Coils having extended linear regions can be made smaller for a given sample size. Among the advantages that can accrue from using smaller coils are improved gradient strength and switching time, reduced eddy currents in the absence of shielding, and improved use of bore space. We used an approximation technique to predict the remaining eddy currents and a time-domain model of coil performance to simulate the electrical performance of the CRP coil and several reduced volume coils of more conventional design. One of the conventional coils was designed based on the time-domain performance model. A single-point acquisition technique was developed to measure the remaining eddy currents of the reduced volume coils. Adaptive sampling increases the dynamic range of the measurement. Measuring only the center of the stimulated echo removes chemical shift and B_0 inhomogeneity effects. The technique was also used to design an inverse filter to remove the eddy current effects in a larger coil set. We added pulsed field gradient and imaging capability to a 7 T commercial spectrometer to perform neuroscience and embryology research and used it in preliminary studies of binary liquid mixtures separating near a critical point. These techniques and coil designs will find application in research areas ranging from functional imaging to NMR microscopy.

High prevalence of Strongyloides stercoralis in school-aged children in a rural highland of north-western Ethiopia: the role of intensive diagnostic work-up.

PubMed

Amor, Aranzazu; Rodriguez, Esperanza; Saugar, José M; Arroyo, Ana; López-Quintana, Beatriz; Abera, Bayeh; Yimer, Mulat; Yizengaw, Endalew; Zewdie, Derejew; Ayehubizu, Zimman; Hailu, Tadesse; Mulu, Wondemagegn; Echazú, Adriana; Krolewieki, Alejandro J; Aparicio, Pilar; Herrador, Zaida; Anegagrie, Melaku; Benito, Agustín

2016-12-01

Soil-transmitted helminthiases (hookworms, Ascaris lumbricoides and Trichuris trichiura) are extremely prevalent in school-aged children living in poor sanitary conditions. Recent epidemiological data suggest that Strongyloides stercoralis is highly unreported. However, accurate data are essential for conducting interventions aimed at introducing control and elimination programmes. We conducted a cross-sectional survey of 396 randomly selected school-aged children in Amhara region in rural area in north-western Ethiopia, to assess the prevalence of S. stercoralis and other intestinal helminths. We examined stools using three techniques: conventional stool concentration; and two S. stercoralis-specific methods, i.e. the Baermann technique and polymerase chain reaction. The diagnostic accuracy of these three methods was then compared. There was an overall prevalence of helminths of 77.5%, with distribution differing according to school setting. Soil-transmitted helminths were recorded in 69.2%. Prevalence of S. stercoralis and hookworm infection was 20.7 and 54.5%, respectively, and co-infection was detected in 16.3% of cases. Schistosoma mansoni had a prevalence of 15.7%. Prevalence of S. stercoralis was shown 3.5% by the conventional method, 12.1% by the Baermann method, and 13.4% by PCR, which thus proved to be the most sensitive. Our results suggest that S. stercoralis could be overlooked and neglected in Ethiopia, if studies of soil-transmitted helminths rely on conventional diagnostic techniques alone. A combination of molecular and stool microscopy techniques yields a significantly higher prevalence. In view of the fact that current control policies for triggering drug administration are based on parasite prevalence levels, a comprehensive diagnostic approach should instead be applied to ensure comprehensive control of helminth infections.

Nanophotonics with Surface Enhanced Coherent Raman Microscopy

NASA Astrophysics Data System (ADS)

Fast, Alexander

Nonlinear nanophotonics is a rapidly developing field of research that aims at detecting and disentangling weak congested optical signatures on the nanoscale. Sub-wavelength field confinement of the local electromagnetic fields and the resulting field enhancement is achieved by utilizing plasmonic near-field antennas. This allows for probing nanoscopic volumes, a property unattainable by conventional far-field microscopy techniques. Combination of plasmonics and nonlinear optical microscopy provides a path to visualizing a small chemical and spatial subset of target molecules within an ensemble. This is achieved while maintaining rapid signal acquisition, which is necessary for capturing biological processes in living systems. Herein, a novel technique, wide-field surface enhanced coherent anti-Stokes Raman scattering (wfSE-CARS) is presented. This technique allows for isolating weak vibrational signals in nanoscopic proximity to the surface by using chemical sensitivity of coherent Raman microspectroscopy (CRM) and field confinement from surface plasmons supported on a thin gold film. Uniform field enhancement over a large field of view, achieved with surface plasmon polaritons (SPP) in wfSE-CARSS, allows for biomolecular imaging demonstrated on extended structures like phospholipid droplets and live cells. Surface selectivity and chemical contrast are achieved at 70 fJ/mum2 incident energy densities, which is over five orders of magnitude lower than used in conventional point scanning CRM. Next, a novel surface sensing imaging technique, local field induced metal emission (LFIME), is introduced. Presence of a sample material at the surface influences the local fields of a thin flat gold film, such that nonlinear fluorescence signal of the metal can be detected in the far-field. Nanoscale nonmetallic, nonfluorescent objects can be imaged with high signal-to-background ratio and diffraction limited lateral resolution using LFIME. Additionally, structure of the extended samples' surfaces can be visualized with a nanoscale axial resolution providing topographic information. Finally, a platform for coherently interrogating single molecules is presented. Single-molecule limit SE-CARS on non-resonant molecules is achieved by means of 3D local field confinement in the nanojunctions between two spherical gold nanoparticles. Localized plasmon resonance of the dimer nanostructure confines the probe volume down to 1 nm3 and provides the local field enhancement necessary to reach single-molecule detection limit. Nonlinear excitation of Raman vibrations in SE-CARS microspectroscopy allows for higher image acquisition rates than in conventionally used single-molecule surface enhanced Raman spectroscopy (SERS). Therefore, data throughput is significantly improved while preserving spectral information despite the presence of the metal. Data simultaneously acquired from hundreds of nanoantennas allows to establish the peak enhancement factor from the observed count rates and define the maximum allowed local-field that preserves the integrity of the antenna. These results are paramount for the future design of time resolved single-molecule studies with multiple pulsed laser excitations, required for single-molecule coherence manipulation and quantum computing.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Ryosuke; Okajima, Takaharu, E-mail: okajima@ist.hokudai.ac.jp

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

TiO2-V2O5 nanocomposites as alternative energy storage substances for photocatalysts.

PubMed

Ngaotrakanwiwat, Pailin; Meeyoo, Vissanu

2012-01-01

TiO2-V2O5 was prepared and evaluated as an energy storage material for photocatalysts with high capacity and initial charging rate. The compound was successfully obtained by sol-gel technique and effects of compound composition and calcination temperature on the energy storage ability were investigated. The synthesized compounds were characterized by means of X-ray powder diffraction (XRD), scanning electron microscopy equipped with energy-dispersive X-ray analysis (SEM-EDX) and transmission electron microscopy (TEM). The results reveals that the compound of Ti:V molar ratio equal to 1:0.11 calcined at 550 degrees C exhibited superior energy storage ability than parent substances and 1.7-times higher capacity and 2.3-times higher initial charging rate compared to WO3, indicating that the compound is a remarkable alternative to conventional energy storage substances.

Tracking liquid in drying colloidal fluids with polarized light microscopy

NASA Astrophysics Data System (ADS)

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

2014-11-01

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

DETECTION OF GYNECOLOGICAL CANCER—Use of Fluorescence Microscopy to Show Nucleic Acids in Malignant Growth

PubMed Central

Von Bertalanffy, Ludwig; Masin, Marianna; Masin, Francis; Kaplan, Leo

1957-01-01

Early detection of malignant lesions of the cervix, a major problem in gynecology, has been made possible in more cases by the development of exfoliative cytology. Mass-screening programs have been impeded, however, by the demands on time and skill of the examiner as posed by conventional techniques. A new method in exfoliative cytology, using fluorescence microscopy, essentially reduces the time of processing as well as of scanning of specimens. Suspicious cells show flaming orange-red fluorescence of the cytoplasm on a black background, impressively distinct from normal cells and giving a warning signal to the examiner. This color reaction is based upon cytochemical changes—namely, the abundance of ribonucleic acid in vividly growing and especially malignant cells. Besides gynecological material, the method is applicable to other forms of malignant disease. ImagesFigure 1.Figure 2.Figure 3.Figure 4.Figure 5.Figure 6. PMID:13460741

Combined scanning transmission electron microscopy tilt- and focal series.

PubMed

Dahmen, Tim; Baudoin, Jean-Pierre; Lupini, Andrew R; Kübel, Christian; Slusallek, Philipp; de Jonge, Niels

2014-04-01

In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt-focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller "missing wedge" artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

PubMed

Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

2015-08-01

Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

Nanoscale imaging of photocurrent enhancement by resonator array photovoltaic coatings.

PubMed

Ha, Dongheon; Yoon, Yohan; Zhitenev, Nikolai B

2018-04-06

Nanoscale surface patterning commonly used to increase absorption of solar cells can adversely impact the open-circuit voltage due to increased surface area and recombination. Here, we demonstrate absorptivity and photocurrent enhancement using silicon dioxide (SiO 2 ) nanosphere arrays on a gallium arsenide (GaAs) solar cell that do not require direct surface patterning. Due to the combined effects of thin-film interference and whispering gallery-like resonances within nanosphere arrays, there is more than 20% enhancement in both absorptivity and photocurrent. To determine the effect of the resonance coupling between nanospheres, we perform a scanning photocurrent microscopy based on a near-field scanning optical microscopy measurement and find a substantial local photocurrent enhancement. The nanosphere-based antireflection coating (ARC), made by the Meyer rod rolling technique, is a scalable and a room-temperature process; and, can replace the conventional thin-film-based ARCs requiring expensive high-temperature vacuum deposition.

Nanoscale imaging of photocurrent enhancement by resonator array photovoltaic coatings

NASA Astrophysics Data System (ADS)

Ha, Dongheon; Yoon, Yohan; Zhitenev, Nikolai B.

2018-04-01

Nanoscale surface patterning commonly used to increase absorption of solar cells can adversely impact the open-circuit voltage due to increased surface area and recombination. Here, we demonstrate absorptivity and photocurrent enhancement using silicon dioxide (SiO2) nanosphere arrays on a gallium arsenide (GaAs) solar cell that do not require direct surface patterning. Due to the combined effects of thin-film interference and whispering gallery-like resonances within nanosphere arrays, there is more than 20% enhancement in both absorptivity and photocurrent. To determine the effect of the resonance coupling between nanospheres, we perform a scanning photocurrent microscopy based on a near-field scanning optical microscopy measurement and find a substantial local photocurrent enhancement. The nanosphere-based antireflection coating (ARC), made by the Meyer rod rolling technique, is a scalable and a room-temperature process; and, can replace the conventional thin-film-based ARCs requiring expensive high-temperature vacuum deposition.

A facile synthesis of zinc oxide/multiwalled carbon nanotube nanocomposite lithium ion battery anodes by sol-gel method

NASA Astrophysics Data System (ADS)

Köse, Hilal; Karaal, Şeyma; Aydın, Ali Osman; Akbulut, Hatem

2015-11-01

Free standing zinc oxide (ZnO) and multiwalled carbon nanotube (MWCNT) nanocomposite materials are prepared by a sol gel technique giving a new high capacity anode material for lithium ion batteries. Free-standing ZnO/MWCNT nanocomposite anodes with two different chelating agent additives, triethanolamine (TEA) and glycerin (GLY), yield different electrochemical performances. Field emission gun scanning electron microscopy (FEG-SEM), energy dispersive X-ray spectrometer (EDS), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) analyses reveal the produced anode electrodes exhibit a unique structure of ZnO coating on the MWCNT surfaces. Li-ion cell assembly using a ZnO/MWCNT/GLY free-standing anode and Li metal cathode possesses the best discharge capacity, remaining as high as 460 mAh g-1 after 100 cycles. This core-shell structured anode can offer increased energy storage and performance over conventional anodes in Li-ion batteries.

Microcapillary imaging of lamina cribrosa in porcine eyes using photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Moothanchery, Mohesh; Chuangsuwanich, Thanadet; Yan, Alvan Tsz Chung; Schmetterer, Leopold; Girard, Michael J. A.; Pramanik, Manojit

2018-02-01

In order to understand the pathophysiology of glaucoma, Lamina cribrosa (LC) perfusion needs to be the subject of thorough investigation. It is currently difficult to obtain high resolution images of the embedded microcapillary network of the LC using conventional imaging techniques. In this study, an optical resolution photoacoustic microscopy (OR-PAM) system was used for imaging lamina cribrosa of an ex vivo porcine eye. Extrinsic contrast agent was used to perfuse the eye via its ciliary arteries. The OR-PAM system have a lateral resolution of 4 μm and an axial resolution of 30 μm. The high resolution system could able resolve a perfused LC microcapillary network to show vascular structure within the LC thickness. OR-PAM could be a promising imaging modality to study the LC perfusion and hence could be used to elucidate the hemodynamic aspect of glaucoma.

Using Laser Ultrasound to Detect Subsurface Defects in Metal Laser Powder Bed Fusion Components

NASA Astrophysics Data System (ADS)

Everton, Sarah; Dickens, Phill; Tuck, Chris; Dutton, Ben

2018-03-01

Laser powder bed fusion offers many advantages over conventional manufacturing methods, such as the integration of multiple parts that can result in significant weight-savings. The increased design freedom that layer-wise manufacture allows has also been seen to enhance component performance at little or no added cost. For such benefits to be realized, however, the material quality must first be assured. Laser ultrasonic testing is a noncontact inspection technique that has been proposed as suitable for in situ monitoring of metal additive manufacturing processes. This article explores the current capability of this technique to detect manufactured, subsurface defects in Ti-6Al-4V samples, ex situ. The results are compared with x-ray computed tomography reconstructions and focus variation microscopy. Although laser ultrasound has been used to identify material discontinuities, further work is required before this technique could be implemented in situ.

Analysis of FIB-induced damage by electron channelling contrast imaging in the SEM.

PubMed

Gutierrez-Urrutia, Ivan

2017-01-01

We have investigated the Ga + ion-damage effect induced by focused ion beam (FIB) milling in a [001] single crystal of a 316 L stainless steel by the electron channelling contrast imaging (ECCI) technique. The influence of FIB milling on the characteristic electron channelling contrast of surface dislocations was analysed. The ECCI approach provides sound estimation of the damage depth produced by FIB milling. For comparison purposes, we have also studied the same milled surface by a conventional electron backscatter diffraction (EBSD) approach. We observe that the ECCI approach provides further insight into the Ga + ion-damage phenomenon than the EBSD technique by direct imaging of FIB artefacts in the scanning electron microscope. We envisage that the ECCI technique may be a convenient tool to optimize the FIB milling settings in applications where the surface crystal defect content is relevant. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Fabrication of lithographically defined optical coupling facets for silicon-on-insulator waveguides by inductively coupled plasma etching

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yap, K.P.; Lamontagne, B.; Delage, A.

2006-05-15

We present a technique to lithographically define and fabricate all required optical facets on a silicon-on-insulator photonic integrated circuit by an inductively coupled plasma etch process. This technique offers 1 {mu}m positioning accuracy of the facets at any location within the chip and eliminates the need of polishing. Facet fabrication consists of two separate steps to ensure sidewall verticality and minimize attack on the end surfaces of the waveguides. Protection of the waveguides by a thermally evaporated aluminum layer before the 40-70 {mu}m deep optical facet etching has been proven essential in assuring the facet smoothness and integrity. Both scanningmore » electron microscopy analysis and optical measurement results show that the quality of the facets prepared by this technique is comparable to the conventional facets prepared by polishing.« less

Intravital multiphoton fluorescence imaging and optical manipulation of spinal cord in mice, using a compact fiber laser system.

PubMed

Oshima, Yusuke; Horiuch, Hideki; Honkura, Naoki; Hikita, Atsuhiko; Ogata, Tadanori; Miura, Hiromasa; Imamura, Takeshi

2014-09-01

Near-infrared ultrafast lasers are widely used for multiphoton excited fluorescence microscopy in living animals. Ti:Sapphire lasers are typically used for multiphoton excitation, but their emission wavelength is restricted below 1,000 nm. The aim of this study is to evaluate the performance of a compact Ytterbium-(Yb-) fiber laser at 1,045 nm for multiphoton excited fluorescence microscopy in spinal cord injury. In this study, we employed a custom-designed microscopy system with a compact Yb-fiber laser and evaluated the performance of this system in in vivo imaging of brain cortex and spinal cord in YFP-H transgenic mice. For in vivo imaging of brain cortex, sharp images of basal dendrites, and pyramidal cells expressing EYFP were successfully captured using the Yb-fiber laser in our microscopy system. We also performed in vivo imaging of axon fibers of spinal cord in the transgenic mice. The obtained images were almost as sharp as those obtained using a conventional ultrafast laser system. In addition, laser ablation and multi-color imaging could be performed simultaneously using the Yb-fiber laser. The high-peak pulse Yb-fiber laser is potentially useful for multimodal bioimaging methods based on a multiphoton excited fluorescence microscopy system that incorporates laser ablation techniques. Our results suggest that microscopy systems of this type could be utilized in studies of neuroscience and clinical use in diagnostics and therapeutic tool for spinal cord injury in the future. © 2014 Wiley Periodicals, Inc.

Comparative evaluation of ibuprofen/beta-cyclodextrin complexes obtained by supercritical carbon dioxide and other conventional methods.

PubMed

Hussein, Khaled; Türk, Michael; Wahl, Martin A

2007-03-01

The preparation of drug/cyclodextrin complexes is a suitable method to improve the dissolution of poor soluble drugs. The efficacy of the Controlled Particle Deposition (CPD) as a new developed method to prepare these complexes in a single stage process using supercritical carbon dioxide is therefore compared with other conventional methods. Ibuprofen/beta-cyclodextrin complexes were prepared with different techniques and characterized using FTIR-ATR spectroscopy, powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). In addition, the influences of the processing technique on the drug content (HPLC) and the dissolution behavior were studied. Employing the CPD-process resulted in a drug content of 2.8+/-0.22 wt.% in the carrier. The material obtained by CPD showed an improved dissolution rate of ibuprofen at pH 5 compared with the pure drug and its physical mixture with beta-cyclodextrin. In addition CPD material displays the highest dissolution (93.5+/- 2.89% after 75 min) compared to material obtained by co-precipitation (61.3 +/-0.52%) or freeze-drying (90.6 +/-2.54%). This study presents the CPD-technique as a well suitable method to prepare a drug/beta-cyclodextrin complex with improved drug dissolution compared to the pure drug and materials obtained by other methods.

Development of large field-of-view two photon microscopy for imaging mouse cortex (Conference Presentation)

NASA Astrophysics Data System (ADS)

Bumstead, Jonathan; Côté, Daniel C.; Culver, Joseph P.

2017-02-01

Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI's) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample. To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction.

PubMed

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-10

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed "digital color fusion microscopy" (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Flexible thermoset towpregs by electrostatic powder fusion coating

NASA Technical Reports Server (NTRS)

Yang, Pei-Hua; Varughese, Babu; Muzzy, John D.

1991-01-01

Thermoset prepregs of expoxy and polyimide have been produced by electrostatic deposition of charged fluidized polymer powders on spread continuous fiber tows. The powders are melted onto the fibers by radiant heating to adhere the polymer to the fiber. This process produces towpreg uniformly and rapidly without imposing severe stresses on the fibers. The towpregs produced by this novel process were consolidated to make unidirectional laminates for mechanical testing. Low void content samples have been made and demonstrated by C-scan and scanning electron microscopy. The mechanical properties of unidirectional laminates are equivalent to composites fabricated by conventional techniques.

Crystallography of ordered colloids using optical microscopy. 2. Divergent-beam technique.

PubMed

Rogers, Richard B; Lagerlöf, K Peter D

2008-04-10

A technique has been developed to extract quantitative crystallographic data from randomly oriented colloidal crystals using a divergent-beam approach. This technique was tested on a series of diverse experimental images of colloidal crystals formed from monodisperse suspensions of sterically stabilized poly-(methyl methacrylate) spheres suspended in organic index-matching solvents. Complete sets of reciprocal lattice basis vectors were extracted in all but one case. When data extraction was successful, results appeared to be accurate to about 1% for lattice parameters and to within approximately 2 degrees for orientation. This approach is easier to implement than a previously developed parallel-beam approach with the drawback that the divergent-beam approach is not as robust in certain situations with random hexagonal close-packed crystals. The two techniques are therefore complimentary to each other, and between them it should be possible to extract quantitative crystallographic data with a conventional optical microscope from any closely index-matched colloidal crystal whose lattice parameters are compatible with visible wavelengths.

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane.

PubMed

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film.

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

PubMed Central

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2011-01-01

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy. PMID:22140560

Front-loaded smear microscopy for the diagnosis of pulmonary TB in Tripoli, Libya.

PubMed

Gammo, Mohamed; Lamaric, Widad; Hadida, Mukhtar; Abuazza, Aida; Askar, Nabil A; Yassin, Mohammed A; Cuevas, Luis E

2013-02-01

Diagnosis of pulmonary TB by conventional smear microscopy requires patient attendance on 2 consecutive days. We investigated whether collecting sputum on-the-spot for smear microscopy on the day the patient presented was as sensitive and specific for diagnosis as the conventional spot-morning-spot scheme. We enrolled 412 adults who presented between January 2009 and October 2010 at the National Centre for TB Control in Tripoli, Libya, with cough of >2 weeks' duration, into a cross-sectional survey collecting four sputum specimens: on-the-spot and Xspot on Day 1; morning and on-the-spot on Day 2. 97 (24%) of 401 culture results were culture positive. Spot-Xspot and spot-morning smear microscopy had, respectively, 65% and 66% sensitivity and 97% and 96% specificity (p > 0.5). Spot-Xspot-morning and Spot-morning-spot smear-microscopy had, respectively, 67% and 66% sensitivity and 96% and 96% specificity (p > 0.5). For the diagnosis of pulmonary TB, the sensitivity and specificity of front-loaded (same-day) smear microscopy is similar to that of the standard smear microscopy scheme.

Nanoprobe studies: Electrical transport in carbon nanotubes and crystal structure of aluminum nitride surfaces

NASA Astrophysics Data System (ADS)

Biswas, Sujit Kumar

Nanoprobes are an extraordinary set of experimental tools that allow fabrication, manipulation, and measurement in nano-scale systems. The primary use of a nanoprobe for imaging tiny objects is supplemented by powerful electrical techniques, namely scanning surface potential microscopy and current sensing atomic force microscopy. They allow us to measure potential, and current in carbon nanotube circuits. Nanoprobes are superior to conventional two- or four-probe measurements because they can provide spatial information of local electronic properties. This makes them highly attractive in studying junctions and contacts with carbon nanotubes. We have studied single-walled carbon nanotube circuits, forming junctions to other nanotubes. The experimental results indicate that these junctions act like potential barriers of about 50 meV that can confine electrons with an effective mass of 0.003 me , within nanotube channels of length 0.5 mum lying in-between two such potential barriers. This leads to quantization of the channel, forming a resonant tunneling structure. We have also found that single-walled nanotubes have phase coherence lengths of the order of 1 mum. This leads to situations where the electron interference effects at scattering centers need to be considered. We have seen direct evidence of this, in the non-linear resistance increase within nanotubes with few defects. Ambipolar transistor behavior was measured in a p-type single-walled nanotube circuit that showed electron injection across the Schottky junction at high positive bias. We have also studied multi-walled carbon nanotube circuits using scanning potential microscopy, and found that a back gate potential can vary the resistance of the channel. Vertical nanotube arrays, suitable for interconnects, were also measured. These hollow multi-walled nanotube channels were about 45 nm in diameter, and 50 mum in length, fabricated in an anodized alumina template. We found that these structures could sustain current densities greater than 105 A/cm2. Conventional use of nanoprobes in imaging aluminum nitride surfaces displayed curious step bunching structures. We have used an innovative analysis technique with which the bulk lattice constant of the crystal was measured to an accuracy of about 4% of X-ray crystallography value of 0.497 nm. In addition, this technique showed that there were regions on the surface that had a larger lattice parameter of 0.64 nm, which we interpreted to be due to a variation in the chemical composition of the surface such as oxide formation. We believe that this technique may prove useful as a study of chemical-composition variations on a surface as well as relaxation of the surface layer.

Characterization and recovery of copper values from discarded slag.

PubMed

Das, Bisweswar; Mishra, Barada Kanta; Angadi, Shivakumar; Pradhan, Siddharth Kumar; Prakash, Sandur; Mohanty, Jayakrushna

2010-06-01

In any copper smelter large quantities of copper slag are discarded as waste material causing space and environmental problems. This discarded slag contains important amounts of metallic values such as copper and iron. The recovery of copper values from an Indian smelter slag that contains 1.53% Cu, 39.8% Fe and 34.65% SiO(2) was the focus of the present study. A complete investigation of the different phases present in the slag has been carried out by means of optical microscopy, Raman spectroscopy, scanning electron microscopy (SEM), and X-ray diffraction (XRD) techniques. It is observed that iron and silica are mostly associated with the fayalite phase whereas copper is present in both oxide and sulfide phases. These oxide and sulfide phases of copper are mostly present within the slag phase and to some extent the slag is also embedded inside the oxide and sulfide phases. The recovery of copper values from the discarded slag has been explored by applying a flotation technique using conventional sodium isopropyl xanthate (SIX) as the collector. The effects of flotation parameters such as pH and collector concentration are investigated. Under optimum flotation conditions, it is possible to achieve 21% Cu with more than 80% recovery.

Optimization of digital image processing to determine quantum dots' height and density from atomic force microscopy.

PubMed

Ruiz, J E; Paciornik, S; Pinto, L D; Ptak, F; Pires, M P; Souza, P L

2018-01-01

An optimized method of digital image processing to interpret quantum dots' height measurements obtained by atomic force microscopy is presented. The method was developed by combining well-known digital image processing techniques and particle recognition algorithms. The properties of quantum dot structures strongly depend on dots' height, among other features. Determination of their height is sensitive to small variations in their digital image processing parameters, which can generate misleading results. Comparing the results obtained with two image processing techniques - a conventional method and the new method proposed herein - with the data obtained by determining the height of quantum dots one by one within a fixed area, showed that the optimized method leads to more accurate results. Moreover, the log-normal distribution, which is often used to represent natural processes, shows a better fit to the quantum dots' height histogram obtained with the proposed method. Finally, the quantum dots' height obtained were used to calculate the predicted photoluminescence peak energies which were compared with the experimental data. Again, a better match was observed when using the proposed method to evaluate the quantum dots' height. Copyright © 2017 Elsevier B.V. All rights reserved.

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

PubMed

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

Scanning thermo-ionic microscopy for probing local electrochemistry at the nanoscale

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eshghinejad, Ahmadreza; Nasr Esfahani, Ehsan; Wang, Peiqi

2016-05-28

Conventional electrochemical characterization techniques based on voltage and current measurements only probe faradaic and capacitive rates in aggregate. In this work we develop a scanning thermo-ionic microscopy (STIM) to probe local electrochemistry at the nanoscale, based on imaging of Vegard strain induced by thermal oscillation. It is demonstrated from both theoretical analysis and experimental validation that the second harmonic response of thermally induced cantilever vibration, associated with thermal expansion, is present in all solids, whereas the fourth harmonic response, caused by local transport of mobile species, is only present in ionic materials. The origin of STIM response is further confirmedmore » by its reduced amplitude with respect to increased contact force, due to the coupling of stress to concentration of ionic species and/or electronic defects. The technique has been applied to probe Sm-doped Ceria and LiFePO{sub 4}, both of which exhibit higher concentrations of mobile species near grain boundaries. The STIM gives us a powerful method to study local electrochemistry with high sensitivity and spatial resolution for a wide range of ionic systems, as well as ability to map local thermomechanical response.« less

Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

NASA Astrophysics Data System (ADS)

Christensen, A. Kent; Lowry, Terry B.

1995-10-01

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about [minus sign]117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at [minus sign]155 to [minus sign]170°C to produce semithin frozen sections (0.5 to 3 [mu]m thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

Nanomorphology of P3HT:PCBM-based absorber layers of organic solar cells after different processing conditions analyzed by low-energy scanning transmission electron microscopy.

PubMed

Pfaff, Marina; Klein, Michael F G; Müller, Erich; Müller, Philipp; Colsmann, Alexander; Lemmer, Uli; Gerthsen, Dagmar

2012-12-01

In this study the nanomorphology of P3HT:PC61BM absorber layers of organic solar cells was studied as a function of the processing parameters and for P3HT with different molecular weight. For this purpose we apply scanning transmission electron microscopy (STEM) at low electron energies in a scanning electron microscope. This method exhibits sensitive material contrast in the high-angle annular dark-field (HAADF) mode, which is well suited to distinguish materials with similar densities and mean atomic numbers. The images taken with low-energy HAADF STEM are compared with conventional transmission electron microscopy and atomic force microscopy images to illustrate the capabilities of the different techniques. For the interpretation of the low-energy HAADF STEM images, a semiempirical equation is used to calculate the image intensities. The experiments show that the nanomorphology of the P3HT:PC61BM blends depends strongly on the molecular weight of the P3HT. Low-molecular-weight P3HT forms rod-like domains during annealing. In contrast, only small globular features are visible in samples containing high-molecular-weight P3HT, which do not change significantly after annealing at 150°C up to 30 min.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

Quantifying structural alterations in Alzheimer's disease brains using quantitative phase imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lee, Moosung; Lee, Eeksung; Jung, JaeHwang; Yu, Hyeonseung; Kim, Kyoohyun; Yoon, Jonghee; Lee, Shinhwa; Jeong, Yong; Park, YongKeun

2017-02-01

Imaging brain tissues is an essential part of neuroscience because understanding brain structure provides relevant information about brain functions and alterations associated with diseases. Magnetic resonance imaging and positron emission tomography exemplify conventional brain imaging tools, but these techniques suffer from low spatial resolution around 100 μm. As a complementary method, histopathology has been utilized with the development of optical microscopy. The traditional method provides the structural information about biological tissues to cellular scales, but relies on labor-intensive staining procedures. With the advances of illumination sources, label-free imaging techniques based on nonlinear interactions, such as multiphoton excitations and Raman scattering, have been applied to molecule-specific histopathology. Nevertheless, these techniques provide limited qualitative information and require a pulsed laser, which is difficult to use for pathologists with no laser training. Here, we present a label-free optical imaging of mouse brain tissues for addressing structural alteration in Alzheimer's disease. To achieve the mesoscopic, unlabeled tissue images with high contrast and sub-micrometer lateral resolution, we employed holographic microscopy and an automated scanning platform. From the acquired hologram of the brain tissues, we could retrieve scattering coefficients and anisotropies according to the modified scattering-phase theorem. This label-free imaging technique enabled direct access to structural information throughout the tissues with a sub-micrometer lateral resolution and presented a unique means to investigate the structural changes in the optical properties of biological tissues.

Sperm preparation: state-of-the-art—physiological aspects and application of advanced sperm preparation methods

PubMed Central

Henkel, Ralf

2012-01-01

For assisted reproduction technologies (ART), numerous techniques were developed to isolate spermatozoa capable of fertilizing oocytes. While early methodologies only focused on isolating viable, motile spermatozoa, with progress of ART, particularly intracytoplasmic sperm injection (ICSI), it became clear that these parameters are insufficient for the identification of the most suitable spermatozoon for fertilization. Conventional sperm preparation techniques, namely, swim-up, density gradient centrifugation and glass wool filtration, are not efficient enough to produce sperm populations free of DNA damage, because these techniques are not physiological and not modeled on the stringent sperm selection processes taking place in the female genital tract. These processes only allow one male germ cell out of tens of millions to fuse with the oocyte. Sites of sperm selection in the female genital tract are the cervix, uterus, uterotubal junction, oviduct, cumulus oophorus and the zona pellucida. Newer strategies of sperm preparation are founded on: (i) morphological assessment by means of ‘motile sperm organelle morphological examination (MSOME)' (ii) electrical charge; and (iii) molecular binding characteristics of the sperm cell. Whereas separation methods based on electrical charge take advantage of the sperm's adherence to a test tube surface or separate in an electrophoresis, molecular binding techniques use Annexin V or hyaluronic acid (HA) as substrates. Techniques in this category are magnet-activated cell sorting, Annexin V-activated glass wool filtration, flow cytometry and picked spermatozoa for ICSI (PICSI) from HA-coated dishes and HA-containing media. Future developments may include Raman microspectrometry, confocal light absorption and scattering spectroscopic microscopy and polarization microscopy. PMID:22138904

Rotation of endosomes demonstrates coordination of molecular motors during axonal transport.

PubMed

Kaplan, Luke; Ierokomos, Athena; Chowdary, Praveen; Bryant, Zev; Cui, Bianxiao

2018-03-01

Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors.

Optofluidic bioimaging platform for quantitative phase imaging of lab on a chip devices using digital holographic microscopy.

PubMed

Pandiyan, Vimal Prabhu; John, Renu

2016-01-20

We propose a versatile 3D phase-imaging microscope platform for real-time imaging of optomicrofluidic devices based on the principle of digital holographic microscopy (DHM). Lab-on-chip microfluidic devices fabricated on transparent polydimethylsiloxane (PDMS) and glass substrates have attained wide popularity in biological sensing applications. However, monitoring, visualization, and characterization of microfluidic devices, microfluidic flows, and the biochemical kinetics happening in these devices is difficult due to the lack of proper techniques for real-time imaging and analysis. The traditional bright-field microscopic techniques fail in imaging applications, as the microfluidic channels and the fluids carrying biological samples are transparent and not visible in bright light. Phase-based microscopy techniques that can image the phase of the microfluidic channel and changes in refractive indices due to the fluids and biological samples present in the channel are ideal for imaging the fluid flow dynamics in a microfluidic channel at high resolutions. This paper demonstrates three-dimensional imaging of a microfluidic device with nanometric depth precisions and high SNR. We demonstrate imaging of microelectrodes of nanometric thickness patterned on glass substrate and the microfluidic channel. Three-dimensional imaging of a transparent PDMS optomicrofluidic channel, fluid flow, and live yeast cell flow in this channel has been demonstrated using DHM. We also quantify the average velocity of fluid flow through the channel. In comparison to any conventional bright-field microscope, the 3D depth information in the images illustrated in this work carry much information about the biological system under observation. The results demonstrated in this paper prove the high potential of DHM in imaging optofluidic devices; detection of pathogens, cells, and bioanalytes on lab-on-chip devices; and in studying microfluidic dynamics in real time based on phase changes.

Microscopy techniques in flavivirus research.

PubMed

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

PubMed

Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

2013-12-24

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

PubMed Central

Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H.; Wouters, Fred S.; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

2013-01-01

We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions. PMID:24324140

Kelvin Probe Force Microscopy in liquid using Electrochemical Force Microscopy

DOE PAGES

Collins, Liam; Jesse, Stephen; Kilpatrick, J.; ...

2015-01-19

Conventional closed loop-Kelvin probe force microscopy (KPFM) has emerged as a powerful technique for probing electric and transport phenomena at the solid-gas interface. The extension of KPFM capabilities to probe electrostatic and electrochemical phenomena at the solid–liquid interface is of interest for a broad range of applications from energy storage to biological systems. However, the operation of KPFM implicitly relies on the presence of a linear lossless dielectric in the probe-sample gap, a condition which is violated for ionically-active liquids (e.g., when diffuse charge dynamics are present). Here, electrostatic and electrochemical measurements are demonstrated in ionically-active (polar isopropanol, milli-Q watermore » and aqueous NaCl) and ionically-inactive (non-polar decane) liquids by electrochemical force microscopy (EcFM), a multidimensional (i.e., bias- and time-resolved) spectroscopy method. In the absence of mobile charges (ambient and non-polar liquids), KPFM and EcFM are both feasible, yielding comparable contact potential difference (CPD) values. In ionically-active liquids, KPFM is not possible and EcFM can be used to measure the dynamic CPD and a rich spectrum of information pertaining to charge screening, ion diffusion, and electrochemical processes (e.g., Faradaic reactions). EcFM measurements conducted in isopropanol and milli-Q water over Au and highly ordered pyrolytic graphite electrodes demonstrate both sample- and solvent-dependent features. Finally, the feasibility of using EcFM as a local force-based mapping technique of material-dependent electrostatic and electrochemical response is investigated. The resultant high dimensional dataset is visualized using a purely statistical approach that does not require a priori physical models, allowing for qualitative mapping of electrostatic and electrochemical material properties at the solid–liquid interface.« less

Mobile microscopy as a screening tool for oral cancer in India: A pilot study

PubMed Central

Skandarajah, Arunan; Sunny, Sumsum P.; Gurpur, Praveen; Reber, Clay D.; D’Ambrosio, Michael V.; Raghavan, Nisheena; James, Bonney Lee; Ramanjinappa, Ravindra D.; Suresh, Amritha; Kandasarma, Uma; Birur, Praveen; Kumar, Vinay V.; Galmeanu, Honorius-Cezar; Itu, Alexandru Mihail; Modiga-Arsu, Mihai; Rausch, Saskia; Sramek, Maria; Kollegal, Manohar; Paladini, Gianluca; Kuriakose, Moni; Koch, Felix; Fletcher, Daniel

2017-01-01

Oral cancer is the most common type of cancer among men in India and other countries in South Asia. Late diagnosis contributes significantly to this mortality, highlighting the need for effective and specific point-of-care diagnostic tools. The same regions with high prevalence of oral cancer have seen extensive growth in mobile phone infrastructure, which enables widespread access to telemedicine services. In this work, we describe the evaluation of an automated tablet-based mobile microscope as an adjunct for telemedicine-based oral cancer screening in India. Brush biopsy, a minimally invasive sampling technique was combined with a simplified staining protocol and a tablet-based mobile microscope to facilitate local collection of digital images and remote evaluation of the images by clinicians. The tablet-based mobile microscope (CellScope device) combines an iPad Mini with collection optics, LED illumination and Bluetooth-controlled motors to scan a slide specimen and capture high-resolution images of stained brush biopsy samples. Researchers at the Mazumdar Shaw Medical Foundation (MSMF) in Bangalore, India used the instrument to collect and send randomly selected images of each slide for telepathology review. Evaluation of the concordance between gold standard histology, conventional microscopy cytology, and remote pathologist review of the images was performed as part of a pilot study of mobile microscopy as a screening tool for oral cancer. Results indicated that the instrument successfully collected images of sufficient quality to enable remote diagnoses that show concordance with existing techniques. Further studies will evaluate the effectiveness of oral cancer screening with mobile microscopy by minimally trained technicians in low-resource settings. PMID:29176904

High-speed laser microsurgery of alert fruit flies for fluorescence imaging of neural activity

PubMed Central

Sinha, Supriyo; Liang, Liang; Ho, Eric T. W.; Urbanek, Karel E.; Luo, Liqun; Baer, Thomas M.; Schnitzer, Mark J.

2013-01-01

Intravital microscopy is a key means of monitoring cellular function in live organisms, but surgical preparation of a live animal for microscopy often is time-consuming, requires considerable skill, and limits experimental throughput. Here we introduce a spatially precise (<1-µm edge precision), high-speed (<1 s), largely automated, and economical protocol for microsurgical preparation of live animals for optical imaging. Using a 193-nm pulsed excimer laser and the fruit fly as a model, we created observation windows (12- to 350-µm diameters) in the exoskeleton. Through these windows we used two-photon microscopy to image odor-evoked Ca2+ signaling in projection neuron dendrites of the antennal lobe and Kenyon cells of the mushroom body. The impact of a laser-cut window on fly health appears to be substantially less than that of conventional manual dissection, for our imaging durations of up to 18 h were ∼5–20 times longer than prior in vivo microscopy studies of hand-dissected flies. This improvement will facilitate studies of numerous questions in neuroscience, such as those regarding neuronal plasticity or learning and memory. As a control, we used phototaxis as an exemplary complex behavior in flies and found that laser microsurgery is sufficiently gentle to leave it intact. To demonstrate that our techniques are applicable to other species, we created microsurgical openings in nematodes, ants, and the mouse cranium. In conjunction with emerging robotic methods for handling and mounting flies or other small organisms, our rapid, precisely controllable, and highly repeatable microsurgical techniques should enable automated, high-throughput preparation of live animals for optical experimentation. PMID:24167298

Spin-coated epoxy resin embedding technique enables facile SEM/FIB thickness determination of porous metal oxide ultra-thin films.

PubMed

Peña, B; Owen, G Rh; Dettelbach, K E; Berlinguette, C P

2018-01-25

A facile nonsubjective method was designed to measure porous nonconductive iron oxide film thickness using a combination of a focused ion beam (FIB) and scanning electron microscopy. Iron oxide films are inherently nonconductive and porous, therefore the objective of this investigation was to optimize a methodology that would increase the conductivity of the film to facilitate high resolution imaging with a scanning electron microscopy and to preserve the porous nature of the film that could potentially be damaged by the energy of the FIB. Sputter coating the sample with a thin layer of iridium before creating the cross section with the FIB decreased sample charging and drifting, but differentiating the iron layer from the iridium coating with backscattered electron imaging was not definitive, making accurate assumptions of the delineation between the two metals difficult. Moreover, the porous nature of the film was lost due to beam damage following the FIB process. A thin layer plastication technique was therefore used to embed the porous film in epoxy resin that would provide support for the film during the FIB process. However, the thickness of the resin created using conventional thin layer plastication processing varied across the sample, making the measuring process only possible in areas where the resin layer was at its thinnest. Such variation required navigating the area for ideal milling areas, which increased the subjectivity of the process. We present a method to create uniform thin resin layers, of controlled thickness, that are ideal for quantifying the thickness of porous nonconductive films with FIB/scanning electron microscopy. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Insights into Comparative Antimicrobial Efficacies of Synthetic and Organic Agents: The Case of ZnS Nanoparticles and Zingiber officinale Rosc.

NASA Astrophysics Data System (ADS)

Obidi, O. F.; Nejo, A. O.; Ayeni, R. A.; Revaprasadu, N.

2018-03-01

The differences among the antimicrobial activities of synthetic nanoparticles (NPs), organic agents and conventional antibiotics against human pathogens are little known. We compared the antimicrobial activities of aqueous, ethanol and ethyl acetate extracts of Zingiber officinale rhizomes with ZnS NPs and tetracycline/nystatin using agar-diffusion techniques. Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and ultraviolet spectroscopy were used to characterize ZnS NPs. At 100 mg/ml, ethanol and ethyl acetate extract inhibited Acinetobacter baumannii, Salmonella typhimurium, Enterococcus faecium, Shigella flexneri, Klebsiella pneumoniae, Staphylococcus epidermidis and Candida albicans with zones of inhibition (ZOI) ranging between 0-42 mm and 0-39 mm, respectively. Candida albicans had a remarkable ZOI of 42 mm and 22 mm from ethanol and ZnS NPs compared with 20 mm from conventional nystatin. TEM and FTIR revealed spherically shaped polydispersed NPs with particle size of 12.5 nm and the role of banana peel extracts in ZnS NPs synthesis. Organic and synthetic NPs proved potential alternatives to conventional antimicrobial agents.

Insights into Comparative Antimicrobial Efficacies of Synthetic and Organic Agents: The Case of ZnS Nanoparticles and Zingiber officinale Rosc.

NASA Astrophysics Data System (ADS)

Obidi, O. F.; Nejo, A. O.; Ayeni, R. A.; Revaprasadu, N.

2018-06-01

The differences among the antimicrobial activities of synthetic nanoparticles (NPs), organic agents and conventional antibiotics against human pathogens are little known. We compared the antimicrobial activities of aqueous, ethanol and ethyl acetate extracts of Zingiber officinale rhizomes with ZnS NPs and tetracycline/nystatin using agar-diffusion techniques. Transmission electron microscopy (TEM), Fourier transform infrared (FTIR) and ultraviolet spectroscopy were used to characterize ZnS NPs. At 100 mg/ml, ethanol and ethyl acetate extract inhibited Acinetobacter baumannii, Salmonella typhimurium, Enterococcus faecium, Shigella flexneri, Klebsiella pneumoniae, Staphylococcus epidermidis and Candida albicans with zones of inhibition (ZOI) ranging between 0-42 mm and 0-39 mm, respectively. Candida albicans had a remarkable ZOI of 42 mm and 22 mm from ethanol and ZnS NPs compared with 20 mm from conventional nystatin. TEM and FTIR revealed spherically shaped polydispersed NPs with particle size of 12.5 nm and the role of banana peel extracts in ZnS NPs synthesis. Organic and synthetic NPs proved potential alternatives to conventional antimicrobial agents.

A microscopy method for scanning transmission electron microscopy imaging of the antibacterial activity of polymeric nanoparticles on a biofilm with an ionic liquid.

PubMed

Takahashi, Chisato; Muto, Shunsuke; Yamamoto, Hiromitsu

2017-08-01

In this study, we developed a scanning transmission electron microscopy (STEM) method for imaging the antibacterial activity of organic polymeric nanoparticles (NPs) toward biofilms formed by Staphylococcus epidermidis bacterial cells, for optimizing NPs to treat biofilm infections. The combination of sample preparation method using a hydrophilic ionic liquid (IL) and STEM observation using the cooling holder eliminates the need for specialized equipment and techniques for biological sample preparation. The annular dark-field STEM results indicated that the two types of biodegradable poly-(DL-lactide-co-glycolide) (PLGA) NPs: PLGA modified with chitosan (CS), and clarithromycin (CAM)-loaded + CS-modified PLGA, prepared by emulsion solvent diffusion exhibited different antibacterial activities in nanoscale. To confirm damage to the sample during STEM observation, we observed the PLGA NPs and the biofilm treated with PLGA NPs by both the conventional method and the newly developed method. The optimized method allows microstructure of the biofilm treated with PLGA NPs to be maintained for 25 min at a current flow of 40 pA. The developed simple sample preparation method would be helpful to understand the interaction of drugs with target materials. In addition, this technique could contribute to the visualization of other deformable composite materials at the nanoscale level. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1432-1437, 2017. © 2016 Wiley Periodicals, Inc.

Two-dimensional directional synthetic aperture focusing technique using acoustic-resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Jeon, Seungwan; Park, Jihoon; Kim, Chulhong

2018-02-01

Photoacoustic microscopy (PAM) is a hybrid imaging technology using optical illumination and acoustic detection. PAM is divided into two types: optical-resolution PAM (OR-PAM) and acoustic-resolution photoacoustic microscopy (AR-PAM). Among them, AR-PAM has a great advantage in the penetration depth compared to OR-PAM because ARPAM relies on the acoustic focus, which is much less scattered in biological tissue than optical focus. However, because the acoustic focus is not as tight as the optical focus with a same numerical aperture (NA), the AR-PAM requires acoustic NA higher than optical NA. The high NA of the acoustic focus produces good image quality in the focal zone, but significantly degrades spatial resolution and signal-to-noise ratio (SNR) in the out-of-focal zone. To overcome the problem, synthetic aperture focusing technique (SAFT) has been introduced. SAFT improves the degraded image quality in terms of both SNR and spatial resolution in the out-of-focus zone by calculating the time delay of the corresponding signals and combining them. To extend the dimension of correction effect, several 2D SAFTs have been introduced, but there was a problem that the conventional 2D SAFTs cannot improve the degraded SNR and resolution as 1D SAFT can do. In this study, we proposed a new 2D SAFT that can compensate the distorted signals in x and y directions while maintaining the correction performance as the 1D SAFT.

Multicellular tumor spheroids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration.

PubMed

Ma, Hui-li; Jiang, Qiao; Han, Siyuan; Wu, Yan; Cui Tomshine, Jin; Wang, Dongliang; Gan, Yaling; Zou, Guozhang; Liang, Xing-Jie

2012-01-01

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.

Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 1; A Study Employing Neural Cell Adhesion Molecules (N-CAM) Immunocytochemistry and Conventional Morphological Techniques (Light and Electron Microscopy)

NASA Technical Reports Server (NTRS)

Daunton, N. G.; DAmelio, F.; Wu, L.; Ilyina-Kakueva, E. I.; Krasnov, I. B.; Hyde, T. M.; Sigworth, S. K.

1994-01-01

The effects of spaceflight upon the 'slow' muscle adductor longus was examined in rats flown in the Soviet Biosatellite COSMOS 2044. Three groups - synchronous, vivarium and basal served as controls. The techniques employed included standard methods for light microscopy, N-CAM immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy, contraction bands and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leucocytes and mononuclear cells. N-CAM immunoreactivity was seen (N-CAM-IR) on the myofiber surface, satellite cells and in regenerating myofibers reminiscent of myotubes. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments that displayed varied distributive patterns. The principal electron microscopic changes of the neuromuscular junctions consisted of a decrease or absence of synaptic vesicles, degeneration of axon terminals, increased number of microtubules, vacant axonal spaces and axonal sprouting. The present observations indicate that major alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Enhanced speed in fluorescence imaging using beat frequency multiplexing

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

A new oxidation based technique for artifact free TEM specimen preparation of nuclear graphite

NASA Astrophysics Data System (ADS)

Johns, Steve; Shin, Wontak; Kane, Joshua J.; Windes, William E.; Ubic, Rick; Karthik, Chinnathambi

2018-07-01

Graphite is a key component in designs of current and future nuclear reactors whose in-service lifetimes are dependent upon the mechanical performance of the graphite. Irradiation damage from fast neutrons creates lattice defects which have a dynamic effect on the microstructure and mechanical properties of graphite. Transmission electron microscopy (TEM) can offer real-time monitoring of the dynamic atomic-level response of graphite subjected to irradiation; however, conventional TEM specimen-preparation techniques, such as argon ion milling itself, damage the graphite specimen and introduce lattice defects. It is impossible to distinguish these defects from the ones created by electron or neutron irradiation. To ensure that TEM specimens are artifact-free, a new oxidation-based technique has been developed. Bulk nuclear grades of graphite (IG-110 and NBG-18) and highly oriented pyrolytic graphite (HOPG) were initially mechanically thinned to ∼60 μm. Discs 3 mm in diameter were then oxidized at temperatures between 575 °C and 625 °C in oxidizing gasses using a new jet-polisher-like set-up in order to achieve optimal oxidation conditions to create self-supporting electron-transparent TEM specimens. The quality of these oxidized specimens were established using optical and electron microscopy. Samples oxidized at 575 °C exhibited large areas of electron transparency and the corresponding lattice imaging showed no apparent damage to the graphite lattice.

A new oxidation based technique for artifact free TEM specimen preparation of nuclear graphite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johns, Steve; Shin, Wontak; Kane, Joshua J.

Graphite is a key component in designs of current and future nuclear reactors whose in-service lifetimes are dependent upon the mechanical performance of the graphite. Irradiation damage from fast neutrons creates lattice defects which have a dynamic effect on the microstructure and mechanical properties of graphite. Transmission electron microscopy (TEM) can offer real-time monitoring of the dynamic atomic-level response of graphite subjected to irradiation; however, conventional TEM specimen-preparation techniques, such as argon ion milling itself, damage the graphite specimen and introduce lattice defects. It is impossible to distinguish these defects from the ones created by electron or neutron irradiation. Thus,tomore » ensure that TEM specimens are artifact-free, a new oxidation-based technique has been developed. Bulk nuclear grades of graphite (IG-110 and NBG-18) and highly oriented pyrolytic graphite (HOPG) were initially mechanically thinned to ~60μm. Discs 3mm in diameter were then oxidized at temperatures between 575°C and 625°C in oxidizing gasses using a new jet-polisher-like set-up in order to achieve optimal oxidation conditions to create self-supporting electron-transparent TEM specimens. The quality of these oxidized specimens were established using optical and electron microscopy. Samples oxidized at 575°C exhibited large areas of electron transparency and the corresponding lattice imaging showed no apparent damage to the graphite lattice.« less

Fixture-abutment connection surface and micro-gap measurements by 3D micro-tomographic technique analysis.

PubMed

Meleo, Deborah; Baggi, Luigi; Di Girolamo, Michele; Di Carlo, Fabio; Pecci, Raffaella; Bedini, Rossella

2012-01-01

X-ray micro-tomography (micro-CT) is a miniaturized form of conventional computed axial tomography (CAT) able to investigate small radio-opaque objects at a-few-microns high resolution, in a non-destructive, non-invasive, and tri-dimensional way. Compared to traditional optical and electron microscopy techniques, which provide two-dimensional images, this innovative investigation technology enables a sample tri-dimensional analysis without cutting, coating or exposing the object to any particular chemical treatment. X-ray micro-tomography matches ideal 3D microscopy features: the possibility of investigating an object in natural conditions and without any preparation or alteration; non-invasive, non-destructive, and sufficiently magnified 3D reconstruction; reliable measurement of numeric data of the internal structure (morphology, structure and ultra-structure). Hence, this technique has multi-fold applications in a wide range of fields, not only in medical and odontostomatologic areas, but also in biomedical engineering, materials science, biology, electronics, geology, archaeology, oil industry, and semi-conductors industry. This study shows possible applications of micro-CT in dental implantology to analyze 3D micro-features of dental implant to abutment interface. Indeed, implant-abutment misfit is known to increase mechanical stress on connection structures and surrounding bone tissue. This condition may cause not only screw preload loss or screw fracture, but also biological issues in peri-implant tissues.

A new oxidation based technique for artifact free TEM specimen preparation of nuclear graphite

DOE PAGES

Johns, Steve; Shin, Wontak; Kane, Joshua J.; ...

2018-04-03

Graphite is a key component in designs of current and future nuclear reactors whose in-service lifetimes are dependent upon the mechanical performance of the graphite. Irradiation damage from fast neutrons creates lattice defects which have a dynamic effect on the microstructure and mechanical properties of graphite. Transmission electron microscopy (TEM) can offer real-time monitoring of the dynamic atomic-level response of graphite subjected to irradiation; however, conventional TEM specimen-preparation techniques, such as argon ion milling itself, damage the graphite specimen and introduce lattice defects. It is impossible to distinguish these defects from the ones created by electron or neutron irradiation. Thus,tomore » ensure that TEM specimens are artifact-free, a new oxidation-based technique has been developed. Bulk nuclear grades of graphite (IG-110 and NBG-18) and highly oriented pyrolytic graphite (HOPG) were initially mechanically thinned to ~60μm. Discs 3mm in diameter were then oxidized at temperatures between 575°C and 625°C in oxidizing gasses using a new jet-polisher-like set-up in order to achieve optimal oxidation conditions to create self-supporting electron-transparent TEM specimens. The quality of these oxidized specimens were established using optical and electron microscopy. Samples oxidized at 575°C exhibited large areas of electron transparency and the corresponding lattice imaging showed no apparent damage to the graphite lattice.« less

Optical sectioning microscopy using two-frame structured illumination and Hilbert-Huang data processing

NASA Astrophysics Data System (ADS)

Trusiak, M.; Patorski, K.; Tkaczyk, T.

2014-12-01

We propose a fast, simple and experimentally robust method for reconstructing background-rejected optically-sectioned microscopic images using two-shot structured illumination approach. Innovative data demodulation technique requires two grid-illumination images mutually phase shifted by π (half a grid period) but precise phase displacement value is not critical. Upon subtraction of the two frames the input pattern with increased grid modulation is computed. The proposed demodulation procedure comprises: (1) two-dimensional data processing based on the enhanced, fast empirical mode decomposition (EFEMD) method for the object spatial frequency selection (noise reduction and bias term removal), and (2) calculating high contrast optically-sectioned image using the two-dimensional spiral Hilbert transform (HS). The proposed algorithm effectiveness is compared with the results obtained for the same input data using conventional structured-illumination (SIM) and HiLo microscopy methods. The input data were collected for studying highly scattering tissue samples in reflectance mode. In comparison with the conventional three-frame SIM technique we need one frame less and no stringent requirement on the exact phase-shift between recorded frames is imposed. The HiLo algorithm outcome is strongly dependent on the set of parameters chosen manually by the operator (cut-off frequencies for low-pass and high-pass filtering and η parameter value for optically-sectioned image reconstruction) whereas the proposed method is parameter-free. Moreover very short processing time required to efficiently demodulate the input pattern predestines proposed method for real-time in-vivo studies. Current implementation completes full processing in 0.25s using medium class PC (Inter i7 2,1 GHz processor and 8 GB RAM). Simple modification employed to extract only first two BIMFs with fixed filter window size results in reducing the computing time to 0.11s (8 frames/s).

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

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.

Electrical Investigation of Nanostructured Fe2O3/p-Si Heterojunction Diode Fabricated Using the Sol-Gel Technique

NASA Astrophysics Data System (ADS)

Mansour, Shehab A.; Ibrahim, Mervat M.

2017-11-01

Iron oxide (α-Fe2O3) nanocrystals have been synthesized via the sol-gel technique. The structural and morphological features of these nanocrystals were studied using x-ray diffraction, Fourier transform-infrared spectroscopy and transmission electron microscopy. Colloidal solution of synthesized α-Fe2O3 (hematite) was spin-coated onto a single-crystal p-type silicon (p-Si) wafer to fabricate a heterojunction diode with Mansourconfiguration Ag/Fe2O3/p-Si/Al. This diode was electrically characterized at room temperature using current-voltage (I-V) characteristics in the voltage range from -9 V to +9 V. The fabricated diode showed a good rectification behavior with a rectification factor 1.115 × 102 at 6 V. The junction parameters such as ideality factor, barrier height, series resistance and shunt resistance are determined using conventional I-V characteristics. For low forward voltage, the conduction mechanism is dominated by the defect-assisted tunneling process with conventional electron-hole recombination. However, at higher voltage, I-V ohmic and space charge-limited current conduction was became less effective with the contribution of the trapped-charge-limited current at the highest voltage range.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Comparative analysis of Trichuris muris surface using conventional, low vacuum, environmental and field emission scanning electron microscopy.

PubMed

Lopes Torres, Eduardo José; de Souza, Wanderley; Miranda, Kildare

2013-09-23

The whipworm of the genus Trichuris Roederer, 1791, is a nematode of worldwide distribution and comprises species that parasitize humans and other mammals. Infections caused by Trichuris spp. in mammals can lead to various intestinal diseases of human and veterinary interest. The morphology of Trichuris spp. and other helminths has been mostly studied using conventional scanning electron microscopy of chemically fixed, dried and metal-coated specimens, although this kind of preparation has been shown to introduce a variety of artifacts such as sample shrinking, loss of secreted products and/or hiding of small structures due to sample coating. Low vacuum (LVSEM) and environmental scanning electron microscopy (ESEM) have been applied to a variety of insulator samples, also used in the visualization of hydrated and/or live specimens in their native state. In the present work, we used LVSEM and ESEM to analyze the surface of T. muris and analyze its interaction with the host tissue using freshly fixed or unfixed hydrated samples. Analysis of hydrated samples showed a set of new features on the surface of the parasite and the host tissue, including the presence of the secretory products of the bacillary glands on the surface of the parasite, and the presence of mucous material and eggs on the intestinal surface. Field emission scanning electron microscopy (FESEM) was also applied to reveal the detailed structure of the glandular chambers in fixed, dried and metal coated samples. Taken together, the results show that analysis of hydrated samples may provide new insights in the structural organization of the surface of helminth parasites and its interaction with the infected tissue, suggesting that the application of alternative SEM techniques may open new perspectives for analysis in taxonomy, morphology and host-parasite interaction fields. Copyright © 2013 Elsevier B.V. All rights reserved.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

PubMed Central

Zhou, Lulu; Cai, Mingjun; Tong, Ti; Wang, Hongda

2017-01-01

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy. PMID:28441775

Correlative Single-Molecule Localization Microscopy and Confocal Microscopy.

PubMed

Soeller, Christian; Hou, Yufeng; Jayasinghe, Isuru D; Baddeley, David; Crossman, David

2017-01-01

Single-molecule localization microscopy allows the ability to image fluorescence labeled molecular targets at nanoscale resolution. However, for many biological questions the ability to provide tissue and cellular context in addition to these high resolution data is eminently informative. Here, we describe a procedure to achieve this aim by correlatively imaging human cardiac tissue first at the nanoscale with direct stochastic optical reconstruction microscopy (dSTORM) and then at the diffraction limit with conventional confocal microscopy.

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

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Cost-utility analysis of LED fluorescence microscopy in the diagnosis of pulmonary tuberculosis in Indian settings.

PubMed

Kelly, V; Sagili, K D; Satyanarayana, S; Reza, L W; Chadha, S S; Wilson, N C

2015-06-01

With support from the Stop TB Partnership's TB REACH Wave 2 Grant, diagnostic microscopy services for tuberculosis (TB) were upgraded from conventional Ziehl-Neelsen (ZN) based sputum microscopy to light emitting diode technology-based fluorescence microscopy (LED FM) in 200 high-workload microscopy centres in India as a pilot intervention. To evaluate the cost-effectiveness of LED-FM over conventional ZN microscopy to inform further scale-up. A decision-tree model was constructed to assess the cost utility of LED FM over ZN microscopy. The results were summarised using incremental cost-effectiveness ratio (ICER); one-way and probabilistic sensitivity analyses were also conducted to address uncertainty within the model. Data were analysed from 200 medical colleges in 2011 and 2012, before and after the introduction of LED microscopes. A full costing analysis was carried out from the perspective of a national TB programme. The ICER was calculated at US$14.64 per disability-adjusted life-year, with an 82% probability of being cost-effective at a willingness-to-pay threshold equivalent to Indian gross domestic product per capita. LED FM is a cost-effective intervention for detecting TB cases in India at high-workload medical college settings.

Development of a HIV-1 Virus Detection System Based on Nanotechnology.

PubMed

Lee, Jin-Ho; Oh, Byung-Keun; Choi, Jeong-Woo

2015-04-27

Development of a sensitive and selective detection system for pathogenic viral agents is essential for medical healthcare from diagnostics to therapeutics. However, conventional detection systems are time consuming, resource-intensive and tedious to perform. Hence, the demand for sensitive and selective detection system for virus are highly increasing. To attain this aim, different aspects and techniques have been applied to develop virus sensor with improved sensitivity and selectivity. Here, among those aspects and techniques, this article reviews HIV virus particle detection systems incorporated with nanotechnology to enhance the sensitivity. This review mainly focused on four different detection system including vertically configured electrical detection based on scanning tunneling microscopy (STM), electrochemical detection based on direct electron transfer in virus, optical detection system based on localized surface plasmon resonance (LSPR) and surface enhanced Raman spectroscopy (SERS) using plasmonic nanoparticle.

A structural investigation of a synthesized precursor for optical fiber applications; the heterobimetallic ErNb 2(OPr i) 13

NASA Astrophysics Data System (ADS)

Engholm, M.; Norin, L.; Edvardsson, S.; Lashgari, K.; Westin, G.

2006-12-01

A structural investigation of a synthesized precursor in a silica glass matrix is performed. Silica soot samples are doped with the heterobimetallic precursor ErNb 2(OPr i) 13 by using a conventional solution doping technique and heat treatments to different temperatures. The precursor has also been introduced into a silica fiber preform by using the modified chemical vapor deposition technique. Analyses are made by using ultraviolet-visible-near infrared absorption spectroscopy, scanning electron microscopy, energy dispersive spectroscopy and powder X-ray diffraction. It is concluded that an immiscible system of ErNbO 4 crystallites and Nb 2O 5 is formed in the silica soot samples at high temperatures. Colloidal particles of ErNbO 4 are also formed in the silica glass fiber preform showing interesting features.

Laser scanning saturated structured illumination microscopy based on phase modulation

NASA Astrophysics Data System (ADS)

Huang, Yujia; Zhu, Dazhao; Jin, Luhong; Kuang, Cuifang; Xu, Yingke; Liu, Xu

2017-08-01

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

PubMed

Ilev, Ilko; Waynant, Ronald; Gannot, Israel; Gandjbakhche, Amir

2007-09-01

A novel fiber-optic confocal approach for ultrahigh depth-resolution (

Microstructure and corrosion resistance of nitrogen-rich surface layers on AISI 304 stainless steel by rapid nitriding in a hollow cathode discharge

NASA Astrophysics Data System (ADS)

Li, Yang; He, Yongyong; Zhang, Shangzhou; Wang, Wei; Zhu, Yijie

2018-01-01

Nitriding treatments have been successfully applied to austenitic stainless steels to improve their hardness and tribological properties. However, at temperatures above 450 °C, conventional plasma nitriding processes decrease the corrosion resistance due to the formation of CrN phases within the modified layer. In this work, AISI 304 austenitic stainless steels were efficiently treated by rapid plasma nitriding at a high temperature of 530 °C in a hollow cathode discharge. The enhanced ionization obtained in the hollow cathode configuration provided a high current density and, consequently, a high temperature could be attained in a short time. The nitrided layers were characterized by X-ray diffraction, scanning electron microscopy, atomic force microscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. The results indicated that the dual-layer structure of the nitrided layer consists of a high-N face-centered cubic structure with a free CrN precipitate outer (top) layer and a nitrogen-expanded austenite S-phase bottom layer. The rapid nitriding-assisted hollow cathode discharge technique permits the use of high temperatures, as high as 530 °C, without promoting degradation in the corrosion resistance of stainless steel.

Crystallization behaviors and seal application of basalt based glass-ceramics

NASA Astrophysics Data System (ADS)

Ateş, A.; Önen, U.; Ercenk, E.; Yılmaz, Ş.

2017-02-01

Basalt based glass-ceramics were prepared by conventional melt-quenching technique and subsequently converted to glass-ceramics by a controlled nucleation and crystallization process. Glass materials were obtained by melt at 1500°C and quenched in cold water. The powder materials were made by milling and spin coating. The powders were applied on the 430 stainless steel interconnector material, and heat treatment was carried out. The interface characteristics between the glass-ceramic layer and interconnector were investigated by using X-ray diffraction analysis (XRD) and scanning electron microscopy (SEM). The results showed that the basalt base glass-ceramic sealant material exhibited promising properties to use for SOFC.

Microcircuit failure analysis using the SEM. [Scanning Electron Microscopes

NASA Technical Reports Server (NTRS)

Nicolas, D. P.

1974-01-01

The scanning electron microscope adds a new dimension to the knowledge that can be obtained from a failed microcircuit. When used with conventional techniques, SEM assists and clarifies the analysis, but it does not replace light microscopy. The most advantageous features for microcircuit analysis are long working distances and great depth of field. Manufacturer related failure modes of microcircuits are metallization defects, poor bonding, surface and particle contamination, and design and fabrication faults. User related failure modes are caused by abuse, such as overstress. The Physics of Failure Procedure followed by the Astrionics Laboratory in failure analysis is described, which is designed to obtain maximum information available from each step.

Phase relations in the system CuMoS

USGS Publications Warehouse

Dawei, H.; Chang, L.L.Y.; Knowles, C.R.

1990-01-01

Phase relations in the system CuMoS were studied in the temperature range 500-1000 ??C by using the conventional sealed, evacuated glass capsule technique. Reflected-light microscopy, X-ray powder diffraction and electron microprobe analysis were used for phase characterization. The chevrel-type phase, CuxMo3S4, is stable above 600??C, and forms equilibrium assemblages with the cubic Cu2S solid solution, copper, molybdenum, Mo2S3 and MoS2. Its solid solution ranges from Cu1.50-2.00Mo3S4 at 700??C to Cu1.22-2.00Mo3S4 at 1000 ??C. ?? 1990.

Enhancement of Curie temperature of barium hexaferrite by dense electronic excitations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Manju; Kashyap, Subhash C.; Gupta, Hem C.

2014-07-15

Curie temperature of polycrystalline barium hexaferrite (BaFe{sub 12}O{sub 19}), prepared by conventional solid state technique, is anomalously and significantly enhanced (by nearly 15%) by energetic heavy ion irradiation (150 MeV, Ag{sup 12+}) at ambient temperature due to dense electronic excitations Moderate fluence (1 × 10{sup 12} ions/cm{sup 2}) induces structural defects giving rise to above enhancement. As established by X-ray diffraction, scanning electron microscopy and Raman studies, higher fluence (1 × 10{sup 13} ions/cm{sup 2}) has structurally transformed the sample to amorphous phase with marginal change in magnetization and Curie temperature.

Defect reduction in overgrown semi-polar (11-22) GaN on a regularly arrayed micro-rod array template

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Y.; Bai, J.; Hou, Y.

2016-02-15

We demonstrate a great improvement in the crystal quality of our semi-polar (11-22) GaN overgrown on regularly arrayed micro-rod templates fabricated using a combination of industry-matched photolithography and dry-etching techniques. As a result of our micro-rod configuration specially designed, an intrinsic issue on the anisotropic growth rate which is a great challenge in conventional overgrowth technique for semi-polar GaN has been resolved. Transmission electron microscopy measurements show a different mechanism of defect reduction from conventional overgrowth techniques and also demonstrate major advantages of our approach. The dislocations existing in the GaN micro-rods are effectively blocked by both a SiO{sub 2}more » mask on the top of each GaN micro-rod and lateral growth along the c-direction, where the growth rate along the c-direction is faster than that along any other direction. Basal stacking faults (BSFs) are also effectively impeded, leading to a distribution of BSF-free regions periodically spaced by BSF regions along the [-1-123] direction, in which high and low BSF density areas further show a periodic distribution along the [1-100] direction. Furthermore, a defect reduction model is proposed for further improvement in the crystalline quality of overgrown (11-22) GaN on sapphire.« less

Comparison of sealer penetration using the EndoVac irrigation system and conventional needle root canal irrigation.

PubMed

Kara Tuncer, Aysun; Unal, Bayram

2014-05-01

The aim of this study was to compare the effect of the EndoVac irrigation system (SybronEndo, Orange, CA) and conventional endodontic needle irrigation on sealer penetration into dentinal tubules. Forty single-rooted, recently extracted human maxillary central incisors were randomly divided into 2 groups according to the irrigation technique used: conventional endodontic needle irrigation and EndoVac irrigation. All teeth were instrumented using the ProFile rotary system (Dentsply Maillefer, Ballaigues, Switzerland) and obturated with gutta-percha and AH Plus sealer (Dentsply DeTrey, Konstanz, Germany) labeled with fluorescent dye. Transverse sections at 1, 3, and 5 mm from the root apex were examined using confocal laser scanning microscopy. The total percentage and maximum depth of sealer penetration were then measured. Mann-Whitney test results showed that EndoVac irrigation resulted in a significantly higher percentage of sealer penetration than conventional irrigation at both the 1- and 3-mm levels (P < .05). However, no difference was found at the 5-mm level. The 5-mm sections in each group showed a significantly higher percentage and maximum depth of sealer penetration than did the 1- and 3-mm sections (P < .05). The EndoVac irrigation system significantly improved the sealer penetration at the 1- to 3-mm level over that of conventional endodontic needle irrigation. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

What is the burden of submicroscopic malaria in pregnancy in central India?

PubMed Central

Singh, Neeru; Bharti, Praveen K; Singh, Mrigendra P; Singh, Rajshree; Yeboah-Antwi, Kojo; Desai, Meghna; Udhayakumar, Venkatachalam; Muniyandi, Malaisamy; Hamer, Davidson H; Wylie, Blair J

2015-01-01

Background: Conventional microscopy underestimates the burden of malarial infection when compared with molecular diagnosis using polymerase chain reaction (PCR)-based methods. Lower density parasitemias serve as a reservoir for infection. We evaluated the prevalence of submicroscopic infections in an area of unstable malarial transmission in India and determined whether these infections negatively impacted maternal or fetal outcomes. Methods: This cross-sectional study (2007–2008) was undertaken in two districts of Chhattisgarh, recruiting women from both antenatal clinics (ANCs) and delivery units (DUs). For ANC/DU subjects, peripheral/placental blood, respectively, was obtained for conventional microscopy and collected onto filter paper for PCR analysis. Results: There were 3425 pregnant women, including 2477 ANC subjects and 948 DU subjects who had both microscopic and PCR samples available. Polymerase chain reaction detected significantly more Plasmodium infections than traditional light microscopy both from peripheral (3.4 vs 1.2%; OR 2.9, 95% confidence intervals (CIs) 1.9–4.5) and placental (4.2 vs 1.7%; OR 2.5, 95% CIs 1.4–4.8) blood samples. Submicroscopic infections were not associated with anemia or severe maternal anemia among ANC or DU participants and were not associated with low birth weight (LBW) among DU participants. In contrast, microscopically detected infections were associated with severe anemia and LBW. Conclusions: In this area of unstable malarial transmission from India, submicroscopic infections did not identify a set of pregnant women at increased risk for anemia or LBW. Until PCR techniques become much less expensive and available as a point of care test for the field setting, its use will be limited for malarial detection. PMID:25627878

Evaluation of Left Ventricular Diastolic Dysfunction with Early Systolic Dysfunction Using Two-Dimensional Speckle Tracking Echocardiography in Canine Heart Failure Model.

PubMed

Wu, Wei-Chun; Ma, Hong; Xie, Rong-Ai; Gao, Li-Jian; Tang, Yue; Wang, Hao

2016-04-01

This study evaluated the role of two-dimensional speckle tracking echocardiography (2DSTE) for predicting left ventricular (LV) diastolic dysfunction in pacing-induced canine heart failure. Pacing systems were implanted in 8 adult mongrel dogs, and continuous rapid right ventricular pacing (RVP, 240 beats/min) was maintained for 2 weeks. The obtained measurements from 2DSTE included global strain rate during early diastole (SRe) and during late diastole (SRa) in the longitudinal (L-SRe, L-SRa), circumferential (C-SRe, C-SRa), and radial directions (R-SRe, R-SRa). Changes in heart morphology were observed by light microscopy and transmission electron microscopy at 2 weeks. The onset of LV diastolic dysfunction with early systolic dysfunction occurred 3 days after RVP initiation. Most of the strain rate imaging indices were altered at 1 or 3 days after RVP onset and continued to worsen until heart failure developed. Light and transmission electron microscopy showed myocardial vacuolar degeneration and mitochondrial swelling in the left ventricular at 2 weeks after RVP onset. Pearson's correlation analysis revealed that parameters of conventional echocardiography and 2DSTE showed moderate correlation with LV pressure parameters, including E/Esep' (r = 0.58, P < 0.01), L-SRe (r = -0.58, P < 0.01), E/L-SRe (r = 0.65, P < 0.01), and R-SRe (r = 0.53, P < 0.01). ROC curves analysis showed that these indices of conventional echocardiography and strain rate imaging could effectively predict LV diastolic dysfunction (area under the curve: E/Esep' 0.78; L-SRe 0.84; E/L-SRe 0.80; R-SRe 0.80). 2DSTE was a sensitive and accurate technique that could be used for predicting LV diastolic dysfunction in canine heart failure model. © 2015, Wiley Periodicals, Inc.

Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

PubMed Central

Wu, Yicong; Ghitani, Alireza; Christensen, Ryan; Santella, Anthony; Du, Zhuo; Rondeau, Gary; Bao, Zhirong; Colón-Ramos, Daniel; Shroff, Hari

2011-01-01

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible. PMID:22006307

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane

PubMed Central

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

ABSTRACT Introduction: Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Methods: Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Results: Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Conclusion: Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film. PMID:28902253

Marginal fit of all-ceramic crowns fabricated using two extraoral CAD/CAM systems in comparison with the conventional technique.

PubMed

Alqahtani, Fawaz

2017-01-01

The purpose of this study was to determine the effect of two extraoral computer-aided design (CAD) and computer-aided manufacturing (CAM) systems, in comparison with conventional techniques, on the marginal fit of monolithic CAD/CAM lithium disilicate ceramic crowns. This is an in vitro interventional study. The study was carried out at the Department of Prosthodontics, School of Dentistry, Prince Sattam Bin Abdul-Aziz University, Saudi Arabia, from December 2015 to April 2016. A marginal gap of 60 lithium disilicate crowns was evaluated by scanning electron microscopy. In total, 20 pressable lithium disilicate (IPS e.max Press [Ivoclar Vivadent]) ceramic crowns were fabricated using the conventional lost-wax technique as a control group. The experimental all-ceramic crowns were produced based on a scan stone model and milled using two extraoral CAD/CAM systems: the Cerec group was fabricated using the Cerec CAD/CAM system, and the Trios group was fabricated using Trios CAD and milled using Wieland Zenotec CAM. One-way analysis of variance (ANOVA) and the Scheffe post hoc test were used for statistical comparison of the groups (α=0.05). The mean (±standard deviation) of the marginal gap of each group was as follows: the Control group was 91.15 (±15.35) µm, the Cerec group was 111.07 (±6.33) µm, and the Trios group was 60.17 (±11.09) µm. One-way ANOVA and the Scheffe post hoc test showed a statistically significant difference in the marginal gap between all groups. It can be concluded from the current study that all-ceramic crowns, fabricated using the CAD/CAM system, show a marginal accuracy that is acceptable in clinical environments. The Trios CAD group displayed the smallest marginal gap.

In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230

Experimental and histological investigations of the bone using two different oscillating osteotomy techniques compared with conventional rotary osteotomy.

PubMed

Heinemann, Friedhelm; Hasan, Istabrak; Kunert-Keil, Christiane; Götz, Werner; Gedrange, Tomas; Spassov, Alexander; Schweppe, Janine; Gredes, Tomasz

2012-03-20

Over the past decade, coinciding with the appearance of a number of new ultrasonic surgical devices, there has been a marked increase in interest in the use of ultrasound in oral surgery and implantology as alternative osteotomy method. The aim of this study was the comparison of the effect of osteotomies performed using ultrasonic surgery (Piezosurgery(®)), sonic surgery SONICflex(®) and the conventional bur method on the heat generation within the bone underneath the osteotomy and light-microscopy observations of the bone at different cutting positions in porcine mandibular segments. It was found that the average heat generated by SONICflex(®) sonic device was close to that by conventional rotary bur (1.54-2.29°C), whereas Piezosurgery(®) showed a high generated heat up to 18.17°C. Histological investigations of the bone matrix adjacent to the defect radius showed intact osteocytes with all three instruments and similar wide damage diameter at the bottom region. SONICflex(®) showed smooth cutting surfaces with minimal damage in the upper defect zone. Finally, presented results showed that sonic surgery performed with SONICflex(®) is an alternative osteotomy method and can be used as an alternative to the conventional bur method. Copyright © 2011 Elsevier GmbH. All rights reserved.

A Novel Microcharacterization Technique in the Measurement of Strain and Orientation Gradient in Advanced Materials

NASA Technical Reports Server (NTRS)

Garmestai, H.; Harris, K.; Lourenco, L.

1997-01-01

Representation of morphology and evolution of the microstructure during processing and their relation to properties requires proper experimental techniques. Residual strains, lattice distortion, and texture (micro-texture) at the interface and the matrix of a layered structure or a functionally gradient material and their variation are among parameters important in materials characterization but hard to measure with present experimental techniques. Current techniques available to measure changes in interred material parameters (residual stress, micro-texture, microplasticity) produce results which are either qualitative or unreliable. This problem becomes even more complicated in the case of a temperature variation. These parameters affect many of the mechanical properties of advanced materials including stress-strain relation, ductility, creep, and fatigue. A review of some novel experimental techniques using recent advances in electron microscopy is presented here to measure internal stress, (micro)texture, interracial strength and (sub)grain formation and realignment. Two of these techniques are combined in the chamber of an Environmental Scanning Electron Microscope to measure strain and orientation gradients in advanced materials. These techniques which include Backscattered Kikuchi Diffractometry (BKD) and Microscopic Strain Field Analysis are used to characterize metallic and intermetallic matrix composites and superplastic materials. These techniques are compared with the more conventional x-ray diffraction and indentation techniques.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-06-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available.

Correlative super-resolution fluorescence microscopy combined with optical coherence microscopy

NASA Astrophysics Data System (ADS)

Kim, Sungho; Kim, Gyeong Tae; Jang, Soohyun; Shim, Sang-Hee; Bae, Sung Chul

2015-03-01

Recent development of super-resolution fluorescence imaging technique such as stochastic optical reconstruction microscopy (STORM) and photoactived localization microscope (PALM) has brought us beyond the diffraction limits. It allows numerous opportunities in biology because vast amount of formerly obscured molecular structures, due to lack of spatial resolution, now can be directly observed. A drawback of fluorescence imaging, however, is that it lacks complete structural information. For this reason, we have developed a super-resolution multimodal imaging system based on STORM and full-field optical coherence microscopy (FF-OCM). FF-OCM is a type of interferometry systems based on a broadband light source and a bulk Michelson interferometer, which provides label-free and non-invasive visualization of biological samples. The integration between the two systems is simple because both systems use a wide-field illumination scheme and a conventional microscope. This combined imaging system gives us both functional information at a molecular level (~20nm) and structural information at the sub-cellular level (~1μm). For thick samples such as tissue slices, while FF-OCM is readily capable of imaging the 3D architecture, STORM suffer from aberrations and high background fluorescence that substantially degrade the resolution. In order to correct the aberrations in thick tissues, we employed an adaptive optics system in the detection path of the STORM microscope. We used our multimodal system to obtain images on brain tissue samples with structural and functional information.

Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

NASA Astrophysics Data System (ADS)

Chandra, Subhash; Morrison, George H.

1995-05-01

The transport of K+, Na+, and Ca2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 [mu]m spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged (13C, 15N) amino acid, -arginine. The labeled amino acid was imaged at mass 28 with a Cs+ primary ion beam as the 28(13C15N)- species. After a 4 h exposure of LLC-PK1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.

Color calibration and fusion of lens-free and mobile-phone microscopy images for high-resolution and accurate color reproduction

PubMed Central

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2016-01-01

Lens-free holographic microscopy can achieve wide-field imaging in a cost-effective and field-portable setup, making it a promising technique for point-of-care and telepathology applications. However, due to relatively narrow-band sources used in holographic microscopy, conventional colorization methods that use images reconstructed at discrete wavelengths, corresponding to e.g., red (R), green (G) and blue (B) channels, are subject to color artifacts. Furthermore, these existing RGB colorization methods do not match the chromatic perception of human vision. Here we present a high-color-fidelity and high-resolution imaging method, termed “digital color fusion microscopy” (DCFM), which fuses a holographic image acquired at a single wavelength with a color-calibrated image taken by a low-magnification lens-based microscope using a wavelet transform-based colorization method. We demonstrate accurate color reproduction of DCFM by imaging stained tissue sections. In particular we show that a lens-free holographic microscope in combination with a cost-effective mobile-phone-based microscope can generate color images of specimens, performing very close to a high numerical-aperture (NA) benchtop microscope that is corrected for color distortions and chromatic aberrations, also matching the chromatic response of human vision. This method can be useful for wide-field imaging needs in telepathology applications and in resource-limited settings, where whole-slide scanning microscopy systems are not available. PMID:27283459

Microstructures and microhardness evolutions of melt-spun Al-8Ni-5Nd-4Si alloy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karakoese, Ercan, E-mail: ekarakose@karatekin.edu.tr; Keskin, Mustafa

2012-03-15

Al-Ni-Nd-Si alloy with nominal composition of Al-8 wt.%Ni-5 wt.%Nd-4 wt.%Si was rapidly solidified by using melt-spinning technique to examine the influence of the cooling rate/conditions on microstructure and mechanical properties. The resulting conventional cast (ingot) and melt-spun ribbons were characterized by X-ray diffraction, optical microscopy, scanning electron microscopy together with energy dispersive spectroscopy, differential scanning calorimetry, differential thermal analysis and Vickers microhardness tester. The ingot alloys consists of four phases namely {alpha}-Al, intermetallic Al{sub 3}Ni, Al{sub 11}Nd{sub 3} and fcc Si. Melt-spun ribbons are completely composed of {alpha}-Al phase. The optical microscopy and scanning electron microscopy results show that themore » microstructures of rapidly solidified ribbons are clearly different from their ingot alloy. The change in microhardness is discussed based on the microstructural observations. - Highlights: Black-Right-Pointing-Pointer Rapid solidification allows a reduction in grain size, extended solid solution ranges. Black-Right-Pointing-Pointer We observed the matrix lattice parameter increases with increasing wheel speed. Black-Right-Pointing-Pointer Melt-spun ribbons consist of partly amorphous phases embedded in crystalline phases. Black-Right-Pointing-Pointer The solidification rate is high enough to retain most of alloying elements in the Al matrix. Black-Right-Pointing-Pointer The rapid solidification has effect on the phase constitution.« less

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

PubMed

Seynhaeve, Ann L B; Ten Hagen, Timo L M

2017-11-01

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Label-free photoacoustic nanoscopy

PubMed Central

Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

2014-01-01

Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

Direct determination of the local Hamaker constant of inorganic surfaces based on scanning force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krajina, Brad A.; Kocherlakota, Lakshmi S.; Overney, René M., E-mail: roverney@u.washington.edu

The energetics involved in the bonding fluctuations between nanometer-sized silicon dioxide (SiO{sub 2}) probes and highly oriented pyrolytic graphite (HOPG) and molybdenum disulfide (MoS{sub 2}) could be quantified directly and locally on the submicron scale via a time-temperature superposition analysis of the lateral forces between scanning force microscopy silicon dioxide probes and inorganic sample surfaces. The so-called “intrinsic friction analysis” (IFA) provided direct access to the Hamaker constants for HOPG and MoS{sub 2}, as well as the control sample, calcium fluoride (CaF{sub 2}). The use of scanning probe enables nanoscopic analysis of bonding fluctuations, thereby overcoming challenges associated with largermore » scale inhomogeneity and surface roughness common to conventional techniques used to determine surface free energies and dielectric properties. A complementary numerical analysis based on optical and electron energy loss spectroscopy and the Lifshitz quantum electrodynamic theory of van der Waals interactions is provided and confirms quantitatively the IFA results.« less

Green synthesis of silver nanoparticles and their application to cotton fabrics.

PubMed

Hebeish, A; El-Bisi, M K; El-Shafei, A

2015-01-01

Herein we present a green synthesis of silver nanoparticles (AgNPs) under the reducing action of sugar and the stabilizing action of polyethylene glycol (PEG). Factors affecting the synthesis notably molecular weight of PEG and concentrations of both sugar and silver nitrate were examined for the sake of optimization. Thus obtained AgNPs were characterized, by ultraviolet-visible (UV-vis) spectra for estimation of AgNPs formation and, Transmission electron microscopy (TEM) for determination of size and shape. AgNPs were applied with 1,2,3,4-butane tetracarboxylic acid (BTCA) and sodium hypophosphite (SHP) as a catalyst to cotton fabric. Application was performed according to the conventional pad-dry-cure technique. The treated fabrics were evaluated via monitoring morphological changes of the fibers using scanning electron microscopy (SEM), tensile strength and crease recovery angles in addition to bioassay of antimicrobial activity of the treated fabrics. Research output disclosed that PEG having molecular weight 2000 is the best among the other PEG used. Copyright © 2014 Elsevier B.V. All rights reserved.

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

PubMed Central

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

2015-01-01

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture. PMID:25767692

Simultaneous multiview capture and fusion improves spatial resolution in wide-field and light-sheet microscopy

PubMed Central

Wu, Yicong; Chandris, Panagiotis; Winter, Peter W.; Kim, Edward Y.; Jaumouillé, Valentin; Kumar, Abhishek; Guo, Min; Leung, Jacqueline M.; Smith, Corey; Rey-Suarez, Ivan; Liu, Huafeng; Waterman, Clare M.; Ramamurthi, Kumaran S.; La Riviere, Patrick J.; Shroff, Hari

2016-01-01

Most fluorescence microscopes are inefficient, collecting only a small fraction of the emitted light at any instant. Besides wasting valuable signal, this inefficiency also reduces spatial resolution and causes imaging volumes to exhibit significant resolution anisotropy. We describe microscopic and computational techniques that address these problems by simultaneously capturing and subsequently fusing and deconvolving multiple specimen views. Unlike previous methods that serially capture multiple views, our approach improves spatial resolution without introducing any additional illumination dose or compromising temporal resolution relative to conventional imaging. When applying our methods to single-view wide-field or dual-view light-sheet microscopy, we achieve a twofold improvement in volumetric resolution (~235 nm × 235 nm × 340 nm) as demonstrated on a variety of samples including microtubules in Toxoplasma gondii, SpoVM in sporulating Bacillus subtilis, and multiple protein distributions and organelles in eukaryotic cells. In every case, spatial resolution is improved with no drawback by harnessing previously unused fluorescence. PMID:27761486

Direct determination of the local Hamaker constant of inorganic surfaces based on scanning force microscopy

NASA Astrophysics Data System (ADS)

Krajina, Brad A.; Kocherlakota, Lakshmi S.; Overney, René M.

2014-10-01

The energetics involved in the bonding fluctuations between nanometer-sized silicon dioxide (SiO2) probes and highly oriented pyrolytic graphite (HOPG) and molybdenum disulfide (MoS2) could be quantified directly and locally on the submicron scale via a time-temperature superposition analysis of the lateral forces between scanning force microscopy silicon dioxide probes and inorganic sample surfaces. The so-called "intrinsic friction analysis" (IFA) provided direct access to the Hamaker constants for HOPG and MoS2, as well as the control sample, calcium fluoride (CaF2). The use of scanning probe enables nanoscopic analysis of bonding fluctuations, thereby overcoming challenges associated with larger scale inhomogeneity and surface roughness common to conventional techniques used to determine surface free energies and dielectric properties. A complementary numerical analysis based on optical and electron energy loss spectroscopy and the Lifshitz quantum electrodynamic theory of van der Waals interactions is provided and confirms quantitatively the IFA results.

Direct determination of the local Hamaker constant of inorganic surfaces based on scanning force microscopy.

PubMed

Krajina, Brad A; Kocherlakota, Lakshmi S; Overney, René M

2014-10-28

The energetics involved in the bonding fluctuations between nanometer-sized silicon dioxide (SiO2) probes and highly oriented pyrolytic graphite (HOPG) and molybdenum disulfide (MoS2) could be quantified directly and locally on the submicron scale via a time-temperature superposition analysis of the lateral forces between scanning force microscopy silicon dioxide probes and inorganic sample surfaces. The so-called "intrinsic friction analysis" (IFA) provided direct access to the Hamaker constants for HOPG and MoS2, as well as the control sample, calcium fluoride (CaF2). The use of scanning probe enables nanoscopic analysis of bonding fluctuations, thereby overcoming challenges associated with larger scale inhomogeneity and surface roughness common to conventional techniques used to determine surface free energies and dielectric properties. A complementary numerical analysis based on optical and electron energy loss spectroscopy and the Lifshitz quantum electrodynamic theory of van der Waals interactions is provided and confirms quantitatively the IFA results.

Self-assembled molecular magnets on patterned silicon substrates: bridging bio-molecules with nanoelectronics.

PubMed

Chang, Chia-Ching; Sun, Kien Wen; Lee, Shang-Fan; Kan, Lou-Sing

2007-04-01

The paper reports the methods of preparing molecular magnets and patterning of the molecules on a semiconductor surface. A highly magnetically aligned metallothionein containing Mn and Cd (Mn,Cd-MT-2) is first synthesized, and the molecules are then placed into nanopores prepared on silicon (001) surfaces using electron beam lithography and reactive ion-etching techniques. We have observed the self-assemble growth of the MT molecules on the patterned Si surface such that the MT molecules have grown into rod or ring type three-dimensional nanostructures, depending on the patterned nanostructures on the surface. We also provide scanning electron microscopy, atomic force microscopy, and magnetic force microscope studies of the molecular nanostructures. This engineered molecule shows molecular magnetization and is biocompatible with conventional semiconductors. These features make Mn,Cd-MT-2 a good candidate for biological applications and sensing sources of new nanodevices. Using molecular self-assembly and topographical patterning of the semiconductor substrate, we can close the gap between bio-molecules and nanoelectronics built into the semiconductor chip.

Atomic characterization of Si nanoclusters embedded in SiO2 by atom probe tomography

PubMed Central

2011-01-01

Silicon nanoclusters are of prime interest for new generation of optoelectronic and microelectronics components. Physical properties (light emission, carrier storage...) of systems using such nanoclusters are strongly dependent on nanostructural characteristics. These characteristics (size, composition, distribution, and interface nature) are until now obtained using conventional high-resolution analytic methods, such as high-resolution transmission electron microscopy, EFTEM, or EELS. In this article, a complementary technique, the atom probe tomography, was used for studying a multilayer (ML) system containing silicon clusters. Such a technique and its analysis give information on the structure at the atomic level and allow obtaining complementary information with respect to other techniques. A description of the different steps for such analysis: sample preparation, atom probe analysis, and data treatment are detailed. An atomic scale description of the Si nanoclusters/SiO2 ML will be fully described. This system is composed of 3.8-nm-thick SiO layers and 4-nm-thick SiO2 layers annealed 1 h at 900°C. PMID:21711666

Virtual substrate method for nanomaterials characterization

PubMed Central

Da, Bo; Liu, Jiangwei; Yamamoto, Mahito; Ueda, Yoshihiro; Watanabe, Kazuyuki; Cuong, Nguyen Thanh; Li, Songlin; Tsukagoshi, Kazuhito; Yoshikawa, Hideki; Iwai, Hideo; Tanuma, Shigeo; Guo, Hongxuan; Gao, Zhaoshun; Sun, Xia; Ding, Zejun

2017-01-01

Characterization techniques available for bulk or thin-film solid-state materials have been extended to substrate-supported nanomaterials, but generally non-quantitatively. This is because the nanomaterial signals are inevitably buried in the signals from the underlying substrate in common reflection-configuration techniques. Here, we propose a virtual substrate method, inspired by the four-point probe technique for resistance measurement as well as the chop-nod method in infrared astronomy, to characterize nanomaterials without the influence of underlying substrate signals from four interrelated measurements. By implementing this method in secondary electron (SE) microscopy, a SE spectrum (white electrons) associated with the reflectivity difference between two different substrates can be tracked and controlled. The SE spectrum is used to quantitatively investigate the covering nanomaterial based on subtle changes in the transmission of the nanomaterial with high efficiency rivalling that of conventional core-level electrons. The virtual substrate method represents a benchmark for surface analysis to provide ‘free-standing' information about supported nanomaterials. PMID:28548114

The Development of Teaching and Learning in Bright-Field Microscopy Technique

ERIC Educational Resources Information Center

Iskandar, Yulita Hanum P.; Mahmud, Nurul Ethika; Wahab, Wan Nor Amilah Wan Abdul; Jamil, Noor Izani Noor; Basir, Nurlida

2013-01-01

E-learning should be pedagogically-driven rather than technologically-driven. The objectives of this study are to develop an interactive learning system in bright-field microscopy technique in order to support students' achievement of their intended learning outcomes. An interactive learning system on bright-field microscopy technique was…

Reliable enumeration of malaria parasites in thick blood films using digital image analysis.

PubMed

Frean, John A

2009-09-23

Quantitation of malaria parasite density is an important component of laboratory diagnosis of malaria. Microscopy of Giemsa-stained thick blood films is the conventional method for parasite enumeration. Accurate and reproducible parasite counts are difficult to achieve, because of inherent technical limitations and human inconsistency. Inaccurate parasite density estimation may have adverse clinical and therapeutic implications for patients, and for endpoints of clinical trials of anti-malarial vaccines or drugs. Digital image analysis provides an opportunity to improve performance of parasite density quantitation. Accurate manual parasite counts were done on 497 images of a range of thick blood films with varying densities of malaria parasites, to establish a uniformly reliable standard against which to assess the digital technique. By utilizing descriptive statistical parameters of parasite size frequency distributions, particle counting algorithms of the digital image analysis programme were semi-automatically adapted to variations in parasite size, shape and staining characteristics, to produce optimum signal/noise ratios. A reliable counting process was developed that requires no operator decisions that might bias the outcome. Digital counts were highly correlated with manual counts for medium to high parasite densities, and slightly less well correlated with conventional counts. At low densities (fewer than 6 parasites per analysed image) signal/noise ratios were compromised and correlation between digital and manual counts was poor. Conventional counts were consistently lower than both digital and manual counts. Using open-access software and avoiding custom programming or any special operator intervention, accurate digital counts were obtained, particularly at high parasite densities that are difficult to count conventionally. The technique is potentially useful for laboratories that routinely perform malaria parasite enumeration. The requirements of a digital microscope camera, personal computer and good quality staining of slides are potentially reasonably easy to meet.

Flash sintering of stoichiometric and hyper-stoichiometric urania

DOE PAGES

Valdez, James Anthony; Byler, Darrin David; Kardoulaki, Erofili; ...

2018-03-29

Flash sintering (FS), a novel fabrication technique belonging to the family of field assisted sintering (FAS) techniques, has been utilized in this study to fabricate uranium dioxide (UO 2) pellets. Stoichiometric (UO 2.00) and hyper-stoichiometric (UO 2.16) pellets were flash sintered at 600 °C within a few (2–3) minutes. This is in sharp contrast to conventional sintering where temperatures hundreds of degrees higher are necessary and the sintering time extends to hours. Relating this in terms of the homologous temperature ratio (T H) for both conditions shows that in the case of flash sintering at 600 °C, T H =more » 0.3 versus T H = 0.6 for conventional sintering at 1600 °C. The highest density achieved for a UO 2.00 pellet was 81% theoretical density (TD) when flash sintered at 600 °C for 184 s at a field of 188 V/cm and a current density of 442 mA/mm 2. For the UO 2.16 pellet, the highest achieved density was 92% TD when flash sintered at 600 °C for 140 s at a field of 188 V/cm and a current density of 632 mA/mm 2. X-ray diffraction (XRD) characterization of the sintered pellets showed the final sintered material to be single cubic fluorite phase. Scanning electron microscopy (SEM) of longitudinal sections revealed non-uniform microstructures with regions of high density where the grain size ranged from 1 to 15 μm. Comparisons between conventionally and flash sintered pellets that achieved equivalent shrinkage strains were also conducted. Lastly, in all cases, the flash sintered pellets achieved similar densification to the conventionally sintered pellets at much lower furnace temperatures and shorter times.« less

Flash sintering of stoichiometric and hyper-stoichiometric urania

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valdez, James Anthony; Byler, Darrin David; Kardoulaki, Erofili

Flash sintering (FS), a novel fabrication technique belonging to the family of field assisted sintering (FAS) techniques, has been utilized in this study to fabricate uranium dioxide (UO 2) pellets. Stoichiometric (UO 2.00) and hyper-stoichiometric (UO 2.16) pellets were flash sintered at 600 °C within a few (2–3) minutes. This is in sharp contrast to conventional sintering where temperatures hundreds of degrees higher are necessary and the sintering time extends to hours. Relating this in terms of the homologous temperature ratio (T H) for both conditions shows that in the case of flash sintering at 600 °C, T H =more » 0.3 versus T H = 0.6 for conventional sintering at 1600 °C. The highest density achieved for a UO 2.00 pellet was 81% theoretical density (TD) when flash sintered at 600 °C for 184 s at a field of 188 V/cm and a current density of 442 mA/mm 2. For the UO 2.16 pellet, the highest achieved density was 92% TD when flash sintered at 600 °C for 140 s at a field of 188 V/cm and a current density of 632 mA/mm 2. X-ray diffraction (XRD) characterization of the sintered pellets showed the final sintered material to be single cubic fluorite phase. Scanning electron microscopy (SEM) of longitudinal sections revealed non-uniform microstructures with regions of high density where the grain size ranged from 1 to 15 μm. Comparisons between conventionally and flash sintered pellets that achieved equivalent shrinkage strains were also conducted. Lastly, in all cases, the flash sintered pellets achieved similar densification to the conventionally sintered pellets at much lower furnace temperatures and shorter times.« less

Flash sintering of stoichiometric and hyper-stoichiometric urania

NASA Astrophysics Data System (ADS)

Valdez, J. A.; Byler, D. D.; Kardoulaki, E.; Francis, J. S. C.; McClellan, K. J.

2018-07-01

Flash sintering (FS), a novel fabrication technique belonging to the family of field assisted sintering (FAS) techniques, has been utilized in this study to fabricate uranium dioxide (UO2) pellets. Stoichiometric (UO2.00) and hyper-stoichiometric (UO2.16) pellets were flash sintered at 600 °C within a few (2-3) minutes. This is in sharp contrast to conventional sintering where temperatures hundreds of degrees higher are necessary and the sintering time extends to hours. Relating this in terms of the homologous temperature ratio (TH) for both conditions shows that in the case of flash sintering at 600 °C, TH = 0.3 versus TH = 0.6 for conventional sintering at 1600 °C. The highest density achieved for a UO2.00 pellet was 81% theoretical density (TD) when flash sintered at 600 °C for 185 s at a field of 188 V/cm and a current density of 442 mA/mm2. For the UO2.16 pellet, the highest achieved density was 91% TD when flash sintered at 600 °C for 123 s at a field of 188 V/cm and a current density of 632 mA/mm2. X-ray diffraction (XRD) characterization of the sintered pellets showed the final sintered material to be single cubic fluorite phase. Scanning electron microscopy (SEM) of longitudinal sections revealed non-uniform microstructures with regions of high density where the grain size ranged from 1 to 15 μm. Comparisons between conventionally and flash sintered pellets that achieved equivalent shrinkage strains were also conducted. In all cases, the flash sintered pellets achieved similar densification to the conventionally sintered pellets at much lower furnace temperatures and shorter times.

Three-dimensional rapid visualization of matrix deformations around angiogenic sprouts (Conference Presentation)

NASA Astrophysics Data System (ADS)

Steuwe, Christian; Vayens, Marie-Mo; Jorge Peñas, Alvaro; Krajnik, Bartosz; Van Oosterwyck, Hans; Roeffaers, Maarten B. J.

2017-02-01

At the cell - extracellular matrix interface, physiologically important traction forces exerted by angiogenic sprouts can be investigated indirectly by mapping the consecutive matrix deformations. In this paper we present an approach to study these forces in three dimensions and with high time resolution. The technique employs lightsheet microscopy, in which a sheet of light is used to illuminate the sample - resulting in z-sectioning capability, superior image recording speed and reduced phototoxicity. For this study, human umbilical vein endothelial cells (HUVEC) are transduced with a LifeAct adenoviral vector to visualize the actin cytoskeleton during live sprouting into a collagen type I hydrogel. The calculation of the matrix deformations is formulated as a B-spline-based 3D non-rigid image registration process that warps the image of beads inside the stressed gel to match the image after stress relaxation. Using this approach we study the role of fast moving actin filaments for filopodia- and tip-cell dynamics in 3D under chemically defined culture conditions such as inhibited acto-myosin force generation. With a time resolution in the range of ten seconds, we find that our technique is at least 20 times faster than conventional traction force microscopy based on confocal imaging. Ultimately, this approach will shed light on rapid mechano-chemical feedback mechanisms important for sprouting angiogenesis.

Thermal and ultrasonic influence in the formation of nanometer scale hydroxyapatite bio-ceramic

PubMed Central

Poinern, GJE; Brundavanam, R; Le, X Thi; Djordjevic, S; Prokic, M; Fawcett, D

2011-01-01

Hydroxyapatite (HAP) is a widely used biocompatible ceramic in many biomedical applications and devices. Currently nanometer-scale forms of HAP are being intensely investigated due to their close similarity to the inorganic mineral component of the natural bone matrix. In this study nano-HAP was prepared via a wet precipitation method using Ca(NO3)2 and KH2PO4 as the main reactants and NH4OH as the precipitator under ultrasonic irradiation. The Ca/P ratio was set at 1.67 and the pH was maintained at 9 during the synthesis process. The influence of the thermal treatment was investigated by using two thermal treatment processes to produce ultrafine nano-HAP powders. In the first heat treatment, a conventional radiant tube furnace was used to produce nano-particles with an average size of approximately 30 nm in diameter, while the second thermal treatment used a microwave-based technique to produce particles with an average diameter of 36 nm. The crystalline structure and morphology of all nanoparticle powders produced were investigated using X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR). Both thermal techniques effectively produced ultrafine powders with similar crystalline structure, morphology and particle sizes. PMID:22114473

Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

2017-02-01

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

Mössbauer, TEM/SAED and XRD investigation on waste dumps of the Valea lui Stan gold mines

NASA Astrophysics Data System (ADS)

Constantinescu, Serban Grigore; Udubasa, Sorin S.; Udubasa, Gheorghe; Kuncser, Victor; Popescu-Pogrion, Nicoleta; Mercioniu, Ionel; Feder, Marcel

2012-03-01

The complementary investigation techniques, Mössbauer spectroscopy, transmission electron microscopy with selected area electron diffraction (TEM/SAED), X-ray diffraction (XRD) have been used to investigate the fate of the Valea lui Stan, Romania, gold-ore nanoscale-minerals during the long time of residence in the waste dumps. The preliminary investigations showed such waste dumps to contain significant amount of metals which cannot be identified by conventional methods. An intense research activity started up in order to evaluate the possibilities to recycle Valea lui Stan waste dumps and to recover metals by chemical or phytoextraction procedures. The waste dumps naturally show different mineral constituents with clay minerals as major phases, observed by XRD-technique. Although the waste dumps materials have whitish-yellowish colours, MÖSSBAUER technique evidences the presence of the finely dispersed iron bearing minerals. The authors are focusing to inspect and analyze Fe-compounds in the samples collected from Valea lui Stan's waste dumps in order to identify the magnetic phases by Mössbauer technique.

Self-sensing cantilevers with integrated conductive coaxial tips for high-resolution electrical scanning probe metrology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haemmerli, Alexandre J.; Pruitt, Beth L., E-mail: pruitt@stanford.edu; Harjee, Nahid

The lateral resolution of many electrical scanning probe techniques is limited by the spatial extent of the electrostatic potential profiles produced by their probes. Conventional unshielded conductive atomic force microscopy probes produce broad potential profiles. Shielded probes could offer higher resolution and easier data interpretation in the study of nanostructures. Electrical scanning probe techniques require a method of locating structures of interest, often by mapping surface topography. As the samples studied with these techniques are often photosensitive, the typical laser measurement of cantilever deflection can excite the sample, causing undesirable changes electrical properties. In this work, we present the design,more » fabrication, and characterization of probes that integrate coaxial tips for spatially sharp potential profiles with piezoresistors for self-contained, electrical displacement sensing. With the apex 100 nm above the sample surface, the electrostatic potential profile produced by our coaxial tips is more than 2 times narrower than that of unshielded tips with no long tails. In a scan bandwidth of 1 Hz–10 kHz, our probes have a displacement resolution of 2.9 Å at 293 K and 79 Å at 2 K, where the low-temperature performance is limited by amplifier noise. We show scanning gate microscopy images of a quantum point contact obtained with our probes, highlighting the improvement to lateral resolution resulting from the coaxial tip.« less

New constraints on deformation processes in serpentinite from sub-micron Raman Spectroscopy and TEM

NASA Astrophysics Data System (ADS)

Smith, S. A. F.; Tarling, M.; Rooney, J. S.; Gordon, K. C.; Viti, C.

2017-12-01

Extensive work has been performed to characterize the mineralogical and mechanical properties of the various serpentine minerals (i.e. antigorite, lizardite, chrysotile, polyhedral and polygonal serpentine). However, correct identification of serpentine minerals is often difficult or impossible using conventional analytical techniques such as optical- and SEM-based microscopy, X-ray diffraction and infrared spectroscopy. Transmission Electron Microscopy (TEM) is the best analytical technique to identify the serpentine minerals, but TEM requires complex sample preparation and typically results in very small analysis areas. Sub-micron confocal Raman spectroscopy mapping of polished thin sections provides a quick and relatively inexpensive way of unambiguously distinguishing the main serpentine minerals within their in-situ microstructural context. The combination of high spatial resolution (with a diffraction-limited system, 366 nm), large-area coverage (up to hundreds of microns in each dimension) and ability to map directly on thin sections allows intricate fault rock textures to be imaged at a sample-scale, which can then form the target of more focused TEM work. The potential of sub-micron Raman Spectroscopy + TEM is illustrated by examining sub-micron-scale mineral intergrowths and deformation textures in scaly serpentinites (e.g. dissolution seams, mineral growth in pressure shadows), serpentinite crack-seal veins and polished fault slip surfaces from a serpentinite-bearing mélange in New Zealand. The microstructural information provided by these techniques has yielded new insights into coseismic dehydration and amorphization processes and the interplay between creep and localised rupture in serpentinite shear zones.

Digital and Conventional Microscopy--Learning Effects Detected through Eye Tracking and the Use of Interactive Whiteboards

ERIC Educational Resources Information Center

Berg, Julia; Jäkel, Lissy; Penzes, Anamarija

2016-01-01

Learning the meaningful use of the microscope is an essential requirement in school curricula. Modern science and medicine is hardly conceivable without the inclusion of microscopy. The number of didactic studies in this area, however, is negligible. Real microscopy is rarely used to gain knowledge in higher school years. Could the understanding…

Implementation of LED fluorescence microscopy for diagnosis of pulmonary and HIV-associated tuberculosis in a hospital setting in Indonesia.

PubMed

Chaidir, Lidya; Parwati, Ida; Annisa, Jessi; Muhsinin, Soni; Meilana, Intan; Alisjahbana, Bachti; van Crevel, Reinout

2013-01-01

Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients. We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard. Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of ≥2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23±0.78 vs 5.82±1.60 minutes, P<0.01), while costs per slide were similar. High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories.

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

PubMed

Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

2016-08-01

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. © 2015 The Author. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of the Royal Microscopical Society.

Single-molecule techniques in biophysics: a review of the progress in methods and applications.

PubMed

Miller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J M; Leake, Mark C

2018-02-01

Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

Single-molecule techniques in biophysics: a review of the progress in methods and applications

NASA Astrophysics Data System (ADS)

Miller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J. M.; Leake, Mark C.

2018-02-01

Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in ‘force spectroscopy’ techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

The internal architecture of dendritic spines revealed by super-resolution imaging: What did we learn so far?

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacGillavry, Harold D., E-mail: h.d.macgillavry@uu.nl; Hoogenraad, Casper C., E-mail: c.hoogenraad@uu.nl

2015-07-15

The molecular architecture of dendritic spines defines the efficiency of signal transmission across excitatory synapses. It is therefore critical to understand the mechanisms that control the dynamic localization of the molecular constituents within spines. However, because of the small scale at which most processes within spines take place, conventional light microscopy techniques are not adequate to provide the necessary level of resolution. Recently, super-resolution imaging techniques have overcome the classical barrier imposed by the diffraction of light, and can now resolve the localization and dynamic behavior of proteins within small compartments with nanometer precision, revolutionizing the study of dendritic spinemore » architecture. Here, we highlight exciting new findings from recent super-resolution studies on neuronal spines, and discuss how these studies revealed important new insights into how protein complexes are assembled and how their dynamic behavior shapes the efficiency of synaptic transmission.« less

Dental caries imaging using hyperspectral stimulated Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Wang, Zi; Zheng, Wei; Jian, Lin; Huang, Zhiwei

2016-03-01

We report the development of a polarization-resolved hyperspectral stimulated Raman scattering (SRS) imaging technique based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of dental caries. In our imaging system, hyperspectral SRS images (512×512 pixels) in both fingerprint region (800-1800 cm-1) and high-wavenumber region (2800-3600 cm-1) are acquired in minutes by scanning the wavelength of OPO output, which is a thousand times faster than conventional confocal micro Raman imaging. SRS spectra variations from normal enamel to caries obtained from the hyperspectral SRS images show the loss of phosphate and carbonate in the carious region. While polarization-resolved SRS images at 959 cm-1 demonstrate that the caries has higher depolarization ratio. Our results demonstrate that the polarization resolved-hyperspectral SRS imaging technique developed allows for rapid identification of the biochemical and structural changes of dental caries.

Cathodic cage plasma deposition of TiN and TiO{sub 2} thin films on silicon substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sousa, Romulo R. M. de; Sato, Patricia S.; Nascente, Pedro A. P., E-mail: nascente@ufscar.br

2015-07-15

Cathodic cage plasma deposition (CCPD) was used for growing titanium nitride (TiN) and titanium dioxide (TiO{sub 2}) thin films on silicon substrates. The main advantages of the CCPD technique are the uniformity, tridimensionality, and high rate of the film deposition that occurs at higher pressures, lower temperatures, and lower treatment times than those used in conventional nitriding treatments. In this work, the influence of the temperature and gas atmosphere upon the characteristics of the deposited films was investigated. The TiN and TiO{sub 2} thin films were characterized by x-ray diffraction, scanning electron microscopy, and Raman spectroscopy to analyze their chemical,more » structural, and morphological characteristics, and the combination of these results indicates that the low-cost CCPD technique can be used to produce even and highly crystalline TiN and TiO{sub 2} films.« less

Wide-field FTIR microscopy using mid-IR pulse shaping

PubMed Central

Serrano, Arnaldo L.; Ghosh, Ayanjeet; Ostrander, Joshua S.; Zanni, Martin T.

2015-01-01

We have developed a new table-top technique for collecting wide-field Fourier transform infrared (FTIR) microscopic images by combining a femtosecond pulse shaper with a mid-IR focal plane array. The pulse shaper scans the delay between a pulse pair extremely rapidly for high signal-to-noise, while also enabling phase control of the individual pulses to under-sample the interferograms and subtract background. Infrared absorption images were collected for a mixture of W(CO)6 or Mn2(CO)10 absorbed polystyrene beads, demonstrating that this technique can spatially resolve chemically distinct species. The images are sub-diffraction limited, as measured with a USAF test target patterned on CaF2 and verified with scalar wave simulations. We also find that refractive, rather than reflective, objectives are preferable for imaging with coherent radiation. We discuss this method with respect to conventional FTIR microscopes. PMID:26191843

Molybdenum oxide nanocolloids prepared by an external field-assisted laser ablation in water

NASA Astrophysics Data System (ADS)

Spadaro, Salvatore; Bonsignore, Martina; Fazio, Enza; Cimino, Francesco; Speciale, Antonio; Trombetta, Domenico; Barreca, Francesco; Saija, Antonina; Neri, Fortunato

2018-01-01

he synthesis of extremely stable molybdenum oxide nanocolloids by pulsed laser ablation was studied. This green technique ensures the formation of contaminant-free nanostructures and the absence of by-products. A focused picosecond pulsed laser beam was used to ablate a solid molybdenum target immersed in deionized water. Molybdenum oxide nearly spherical nanoparticles with dimensions within few nanometers (20-100 nm) are synthesized when the ablation processes were carried out, in water, at room temperature and 80°C. The application of an external electric field during the ablation process induces a nanostructures reorganization, as indicated by Scanning-Transmission Electron Microscopy images analysis. The ablation products were also characterized by some spectroscopic techniques: conventional UV-vis optical absorption, atomic absorption, dynamic light scattering, micro-Raman and X-ray photoelectron spectroscopies. Finally, NIH/3T3 mouse fibroblasts were used to evaluate cell viability by the sulforhodamine B assay

Photogrammetry of the three-dimensional shape and texture of a nanoscale particle using scanning electron microscopy and free software.

PubMed

Gontard, Lionel C; Schierholz, Roland; Yu, Shicheng; Cintas, Jesús; Dunin-Borkowski, Rafal E

2016-10-01

We apply photogrammetry in a scanning electron microscope (SEM) to study the three-dimensional shape and surface texture of a nanoscale LiTi2(PO4)3 particle. We highlight the fact that the technique can be applied non-invasively in any SEM using free software (freeware) and does not require special sample preparation. Three-dimensional information is obtained in the form of a surface mesh, with the texture of the sample stored as a separate two-dimensional image (referred to as a UV Map). The mesh can be used to measure parameters such as surface area, volume, moment of inertia and center of mass, while the UV map can be used to study the surface texture using conventional image processing techniques. We also illustrate the use of 3D printing to visualize the reconstructed model. Copyright © 2016 Elsevier B.V. All rights reserved.

Relation between hardness and ultrasonic velocity on pipeline steel welded joints

NASA Astrophysics Data System (ADS)

Carreón, H.; Barrera, G.; Natividad, C.; Salazar, M.; Contreras, A.

2016-04-01

In general, the ultrasonic techniques have been used to determine the mechanical properties of materials based on their relationship with metallurgical characteristics. In this research work, the relationship between ultrasonic wave velocity, hardness and the microstructure of steel pipeline welded joints is investigated. Measurements of ultrasonic wave velocity were made as a function of the location across the weld. Hardness measurements were performed in an attempt to correlate with ultrasonic response. In addition, the coarse and dendritic grain structure of the weld material is extreme and unpredictably anisotropic. Thus, due to the acoustic anisotropy of the crystal, weld material of studied joints is anisotropic too. Such structure is no longer direction-independent to the ultrasonic wave propagation; therefore, the ultrasonic beam deflects and redirects and the wave front becomes distorted. Thus, the use of conventional ultrasonic testing techniques using fixed beam angles is very limited and the application of conventional ultrasonic phased array techniques becomes desirable. This technique is proposed to assist pipeline operators in estimating the hardness through ultrasonic measures to evaluate the susceptibility to stress sulphide cracking and hydrogen-induced cracking due to hard spots in steel pipeline welded joints in service. Sound wave velocity and hardness measurements have been carried out on a steel welded joint. For each section of the welding, weld bead, fusion zone, heat affected zone and base metal were found to correspond particular values of the ultrasound velocity. These results were correlated with electron microscopy observations of the microstructure and sectorial scan view of welded joints by ultrasonic phased array.

Rotation of endosomes demonstrates coordination of molecular motors during axonal transport

PubMed Central

Kaplan, Luke; Ierokomos, Athena; Chowdary, Praveen; Bryant, Zev; Cui, Bianxiao

2018-01-01

Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors. PMID:29536037

Plant cell wall characterization using scanning probe microscopy techniques

PubMed Central

Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

2009-01-01

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

Partial wave spectroscopic microscopy can predict prostate cancer progression and mitigate over-treatment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhang, Di; Graff, Taylor; Crawford, Susan; Subramanian, Hariharan; Thompson, Sebastian; Derbas, Justin R.; Lyengar, Radha; Roy, Hemant K.; Brendler, Charles B.; Backman, Vadim

2016-02-01

Prostate Cancer (PC) is the second leading cause of cancer deaths in American men. While prostate specific antigen (PSA) test has been widely used for screening PC, >60% of the PSA detected cancers are indolent, leading to unnecessary clinical interventions. An alternative approach, active surveillance (AS), also suffer from high expense, discomfort and complications associated with repeat biopsies (every 1-3 years), limiting its acceptance. Hence, a technique that can differentiate indolent from aggressive PC would attenuate the harms from over-treatment. Combining microscopy with spectroscopy, our group has developed partial wave spectroscopic (PWS) microscopy, which can quantify intracellular nanoscale organizations (e.g. chromatin structures) that are not accessible by conventional microscopy. PWS microscopy has previously been shown to predict the risk of cancer in seven different organs (N ~ 800 patients). Herein we use PWS measurement of label-free histologically-normal prostatic epithelium to distinguish indolent from aggressive PC and predict PC risk. Our results from 38 men with low-grade PC indicated that there is a significant increase in progressors compared to non-progressors (p=0.002, effect size=110%, AUC=0.80, sensitivity=88% and specificity=72%), while the baseline clinical characteristics were not significantly different. We further improved the diagnostic power by performing nuclei-specific measurements using an automated system that separates in real-time the cell nuclei from the remaining prostate epithelium. In the long term, we envision that the PWS based prognostication can be coupled with AS without any change to the current procedure to mitigate the harms caused by over-treatment.

Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM).

PubMed

Bridier, A; Meylheuc, T; Briandet, R

2013-05-01

In this contribution, we used a set of microscopic techniques including confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM) and field emission scanning electron microscopy (FESEM) to analyze the three-dimensional spatial arrangement of cells and their surrounding matrix in Bacillus subtilis biofilm. The combination of the different techniques enabled a deeper and realistic deciphering of biofilm architecture by providing the opportunity to overcome the limits of each single technique. Copyright © 2013 Elsevier Ltd. All rights reserved.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Cast and 3D printed ion exchange membranes for monolithic microbial fuel cell fabrication

NASA Astrophysics Data System (ADS)

Philamore, Hemma; Rossiter, Jonathan; Walters, Peter; Winfield, Jonathan; Ieropoulos, Ioannis

2015-09-01

We present novel solutions to a key challenge in microbial fuel cell (MFC) technology; greater power density through increased relative surface area of the ion exchange membrane that separates the anode and cathode electrodes. The first use of a 3D printed polymer and a cast latex membrane are compared to a conventionally used cation exchange membrane. These new techniques significantly expand the geometric versatility available to ion exchange membranes in MFCs, which may be instrumental in answering challenges in the design of MFCs including miniaturisation, cost and ease of fabrication. Under electrical load conditions selected for optimal power transfer, peak power production (mean 10 batch feeds) was 11.39 μW (CEM), 10.51 μW (latex) and 0.92 μW (Tangoplus). Change in conductivity and pH of anolyte were correlated with MFC power production. Digital and environmental scanning electron microscopy show structural changes to and biological precipitation on membrane materials following long term use in an MFC. The cost of the novel membranes was lower than the conventional CEM. The efficacy of two novel membranes for ion exchange indicates that further characterisation of these materials and their fabrication techniques, shows great potential to significantly increase the range and type of MFCs that can be produced.

Mobile Phone Microscopy for the Diagnosis of Soil-Transmitted Helminth Infections: A Proof-of-Concept Study

PubMed Central

Bogoch, Isaac I.; Andrews, Jason R.; Speich, Benjamin; Utzinger, Jürg; Ame, Shaali M.; Ali, Said M.; Keiser, Jennifer

2013-01-01

We created a mobile phone microscope and assessed its accuracy for the diagnosis of soil-transmitted helminths compared with conventional microscopy. Mobile phone microscopy has a sensitivity of 69.4% for detecting any helminth egg and sensitivities of 81.0%, 54.4%, and 14.3% for the diagnosis of Ascaris lumbricoides, Trichuris trichiura and hookworm respectively. PMID:23478580

Laser Surface Preparation for Adhesive Bonding of Ti-6Al-4V

NASA Technical Reports Server (NTRS)

Belcher, Marcus A.; List, Martina S.; Wohl, Christopher J.; Ghose, Sayata; Watson, Kent A.; Hopkins, John W.; Connell, John W.

2010-01-01

Adhesively bonded structures are potentially lighter in weight than mechanically fastened ones, but existing surface treatments are often considered unreliable. Two main problems in achieving reproducible and durable adhesive bonds are surface contamination and variability in standard surface preparation techniques. In this work three surface pretreatments were compared: laser etching with and without grit blasting and conventional Pasa-Jell treatment. Ti-6Al-4V surfaces were characterized by contact angle goniometry, optical microscopy, and X-ray photoelectron spectroscopy (XPS). Laser -etching was found to produce clean surfaces with precisely controlled surface topographies and PETI-5 lap shear strengths and durabilities were equivalent to those produced with Pasa-Jell.

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Effects of the change in cutoff values for human epidermal growth factor receptor 2 status by immunohistochemistry and fluorescence in situ hybridization: a study comparing conventional brightfield microscopy, image analysis-assisted microscopy, and interobserver variation.

PubMed

Atkinson, Roscoe; Mollerup, Jens; Laenkholm, Anne-Vibeke; Verardo, Mark; Hawes, Debra; Commins, Deborah; Engvad, Birte; Correa, Adrian; Ehlers, Charlotte Cort; Nielsen, Kirsten Vang

2011-08-01

New guidelines for HER2 testing have been introduced. To evaluate the difference in HER2 assessment after introduction of new cutoff levels for both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and to compare interobserver agreement and time to score between image analysis and conventional microscopy. Samples from 150 patients with breast cancer were scored by 7 pathologists using conventional microscopy, with a cutoff of both 10% and 30% IHC-stained cells, and using automated microscopy with image analysis. The IHC results were compared individually and to HER2 status as determined by FISH, using both the approved cutoff of 2.0 and the recently introduced cutoff of 2.2. High concordance was found in IHC scoring among the 7 pathologists. The 30% cutoff led to slightly fewer positive IHC observations. Introduction of a FISH equivocal zone affected 4% of the FISH scores. If cutoff for FISH is kept at 2.0, no difference in patient selection is found between the 10% and the 30% IHC cutoff. Among the 150 breast cancer samples, the new 30% IHC and 2.2 FISH cutoff levels resulted in one case without a firm diagnosis because both IHC and FISH were equivocal. Automated microscopy and image analysis-assisted IHC led to significantly better interobserver agreement among the 7 pathologists, with an increase in mean scoring time of only about 30 seconds per slide. The change in cutoff levels led to a higher concordance between IHC and FISH, but fewer samples were classified as HER2 positive.

Ferrous sulfate based low temperature synthesis and magnetic properties of nickel ferrite nanostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tejabhiram, Y., E-mail: tejabhiram@gmail.com; Pradeep, R.; Helen, A.T.

2014-12-15

Highlights: • Novel low temperature synthesis of nickel ferrite nanoparticles. • Comparison with two conventional synthesis techniques including hydrothermal method. • XRD results confirm the formation of crystalline nickel ferrites at 110 °C. • Superparamagnetic particles with applications in drug delivery and hyperthermia. • Magnetic properties superior to conventional methods found in new process. - Abstract: We report a simple, low temperature and surfactant free co-precipitation method for the preparation of nickel ferrite nanostructures using ferrous sulfate as the iron precursor. The products obtained from this method were compared for their physical properties with nickel ferrites produced through conventional co-precipitationmore » and hydrothermal methods which used ferric nitrate as the iron precursor. X-ray diffraction analysis confirmed the synthesis of single phase inverse spinel nanocrystalline nickel ferrites at temperature as low as 110 °C in the low temperature method. Electron microscopy analysis on the samples revealed the formation of nearly spherical nanostructures in the size range of 20–30 nm which are comparable to other conventional methods. Vibrating sample magnetometer measurements showed the formation of superparamagnetic particles with high magnetic saturation 41.3 emu/g which corresponds well with conventional synthesis methods. The spontaneous synthesis of the nickel ferrite nanoparticles by the low temperature synthesis method was attributed to the presence of 0.808 kJ mol{sup −1} of excess Gibbs free energy due to ferrous sulfate precursor.« less

The use of atomic force microscopy to evaluate warm mix asphalt.

DOT National Transportation Integrated Search

2013-01-01

The main objective of this study was to use the Atomic Force Microscopy (AFM) to examine the moisture susceptibility : and healing characteristics of Warm Mix Asphalt (WMA) and compare it with those of conventional Hot Mix Asphalt (HMA). To : this en...

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Digital holographic microscopy applied to measurement of a flow in a T-shaped micromixer

NASA Astrophysics Data System (ADS)

Ooms, T. A.; Lindken, R.; Westerweel, J.

2009-12-01

In this paper, we describe measurements of a three-dimensional (3D) flow in a T-shaped micromixer by means of digital holographic microscopy. Imaging tracer particles in a microscopic flow with conventional microscopy is accompanied by a small depth-of-field, which hinders true volumetric flow measurements. In holographic microscopy, the depth of the measurement domain does not have this limitation because any desired image plane can be reconstructed after recording. Our digital holographic microscope (DHM) consists of a conventional in-line recording system with an added magnifying optical element. The measured flow velocity and the calculated vorticity illustrate four streamwise vortices in the micromixer outflow channel. Because the investigated flow is stationary and strongly 3D, the DHM performance (i.e. accuracy and resolution) can be precisely investigated. The obtained Dynamic spatial range and Dynamic velocity range are larger than 20 and 30, respectively. High-speed multiple-frame measurements illustrate the capability to simultaneously track about 80 particles in a volumetric measurement domain.

Characterization and differentiation of rock varnish types from different environments by microanalytical techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macholdt, D. S.; Jochum, K. P.; Pöhlker, C.

We investigated rock varnishes collected from several locations and environments worldwide by a wide range of microanalytical techniques. These techniques were selected to address the challenges posed by the chemical and structural complexity within the micrometer- to nanometer-sized structures in these geological materials. Femtosecond laser ablation-inductively coupled plasma-mass spectrometry (fs LA-ICP-MS), scanning transmission X-ray microscopy-near edge X-ray adsorption fine structure spectroscopy (STXM-NEXAFS) in combination with scanning electron microscopy (SEM) of focused ion beam (FIB) ultra-thin (100–200 nm) sections, conventional and polarization microscopy, as well as electron paramagnetic resonance (EPR) measurements were used to obtain information about these rock varnishes. Rockmore » varnishes from different environments, which cannot readily be distinguished based on their macroscopic appearance, differ significantly in their constituent elemental mass fractions, e.g., of Mn, Fe, Ni, Co, Ba, and Pb, and their rare earth element (REE) patterns. Structural characteristics such as the particle sizes of embedded dust grains, internal structures such as layers of Mn-, Fe-, and Ca -rich material, and structures such as cavities varied between varnishes from different environments and regions in the world. The EPR spectra were consistent with aged biogenic Mn oxides in all samples, but showed subtle differences between samples of different origin. Our observations allow us to separate rock varnishes into different types, with differences that might be indicators of distinct geneses. Five different types of rock varnish could be distinguished, Type I–V, of which only Type I might be used as potential paleoclimate archive. Each varnish type has specific characteristics in terms of their elemental composition, element distribution, and structures. The combination of element ratios (Mn/Ba, Al/Ni, Mn/REY, Mn/Ce, Mn/Pb, La N /Yb N , and Ce/Ce*), total REE contents, and structures can be used to separate the different types of rock varnish from each other.« less

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

PubMed Central

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M.; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-01-01

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging. PMID:23401517

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging.

PubMed

Shimozawa, Togo; Yamagata, Kazuo; Kondo, Takefumi; Hayashi, Shigeo; Shitamukai, Atsunori; Konno, Daijiro; Matsuzaki, Fumio; Takayama, Jun; Onami, Shuichi; Nakayama, Hiroshi; Kosugi, Yasuhito; Watanabe, Tomonobu M; Fujita, Katsumasa; Mimori-Kiyosue, Yuko

2013-02-26

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

PubMed

Agarwal, Nitin; Biancardi, Alberto M; Patten, Florence W; Reeves, Anthony P; Seibel, Eric J

2014-04-01

Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

Accuracy of Mobile Phone and Handheld Light Microscopy for the Diagnosis of Schistosomiasis and Intestinal Protozoa Infections in Côte d'Ivoire.

PubMed

Coulibaly, Jean T; Ouattara, Mamadou; D'Ambrosio, Michael V; Fletcher, Daniel A; Keiser, Jennifer; Utzinger, Jürg; N'Goran, Eliézer K; Andrews, Jason R; Bogoch, Isaac I

2016-06-01

Handheld light microscopy using compact optics and mobile phones may improve the quality of health care in resource-constrained settings by enabling access to prompt and accurate diagnosis. Laboratory technicians were trained to operate two handheld diagnostic devices (Newton Nm1 microscope and a clip-on version of the mobile phone-based CellScope). The accuracy of these devices was compared to conventional light microscopy for the diagnosis of Schistosoma haematobium, S. mansoni, and intestinal protozoa infection in a community-based survey in rural Côte d'Ivoire. One slide of 10 ml filtered urine and a single Kato-Katz thick smear from 226 individuals were subjected to the Newton Nm1 microscope and CellScope for detection of Schistosoma eggs and compared to conventional microscopy. Additionally, 121 sodium acetate-acetic acid-formalin (SAF)-fixed stool samples were examined by the Newton Nm1 microscope and compared to conventional microscopy for the diagnosis of intestinal protozoa. The prevalence of S. haematobium, S. mansoni, Giardia intestinalis, and Entamoeba histolytica/E. dispar, as determined by conventional microscopy, was 39.8%, 5.3%, 20.7%, and 4.9%, respectively. The Newton Nm1 microscope had diagnostic sensitivities for S. mansoni and S. haematobium infection of 91.7% (95% confidence interval (CI) 59.8-99.6%) and 81.1% (95% CI 71.2-88.3%), respectively, and specificities of 99.5% (95% CI 97.0-100%) and 97.1% (95% CI 92.2-99.1%), respectively. The CellScope demonstrated sensitivities for S. mansoni and S. haematobium of 50.0% (95% CI 25.4-74.6%) and 35.6% (95% CI 25.9-46.4%), respectively, and specificities of 99.5% (95% CI 97.0-100%) and 100% (95% CI 86.7-100%), respectively. For G. intestinalis and E. histolytica/E. dispar, the Newton Nm1 microscope had sensitivity of 84.0% (95% CI 63.1-94.7%) and 83.3% (95% CI 36.5-99.1%), respectively, and 100% specificity. Handheld diagnostic devices can be employed in community-based surveys in resource-constrained settings after minimal training of laboratory technicians to diagnose intestinal parasites.

Web-Based Virtual Microscopy for Parasitology: A Novel Tool for Education and Quality Assurance

PubMed Central

Linder, Ewert; Lundin, Mikael; Thors, Cecilia; Lebbad, Marianne; Winiecka-Krusnell, Jadwiga; Helin, Heikki; Leiva, Byron; Isola, Jorma; Lundin, Johan

2008-01-01

Background The basis for correctly assessing the burden of parasitic infections and the effects of interventions relies on a somewhat shaky foundation as long as we do not know how reliable the reported laboratory findings are. Thus virtual microscopy, successfully introduced as a histopathology tool, has been adapted for medical parasitology. Methodology/Principal Findings Specimens containing parasites in tissues, stools, and blood have been digitized and made accessible as a “webmicroscope for parasitology” (WMP) on the Internet (http://www.webmicroscope.net/parasitology).These digitized specimens can be viewed (“navigated” both in the x-axis and the y-axis) at the desired magnification by an unrestricted number of individuals simultaneously. For virtual microscopy of specimens containing stool parasites, it was necessary to develop the technique further in order to enable navigation in the z plane (i.e., “focusing”). Specimens were therefore scanned and photographed in two or more focal planes. The resulting digitized specimens consist of stacks of laterally “stiched” individual images covering the entire area of the sample photographed at high magnification. The digitized image information (∼10 GB uncompressed data per specimen) is accessible at data transfer speeds from 2 to 10 Mb/s via a network of five image servers located in different parts of Europe. Image streaming and rapid data transfer to an ordinary personal computer makes web-based virtual microscopy similar to conventional microscopy. Conclusion/Significance The potential of this novel technique in the field of medical parasitology to share identical parasitological specimens means that we can provide a “gold standard”, which can overcome several problems encountered in quality control of diagnostic parasitology. Thus, the WMP may have an impact on the reliability of data, which constitute the basis for our understanding of the vast problem of neglected tropical diseases. The WMP can be used also in the absence of a fast Internet communication. An ordinary PC, or even a laptop, may function as a local image server, e.g., in health centers in tropical endemic areas. PMID:18941514

Conventions and workflows for using Situs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wriggers, Willy, E-mail: wriggers@biomachina.org

2012-04-01

Recent developments of the Situs software suite for multi-scale modeling are reviewed. Typical workflows and conventions encountered during processing of biophysical data from electron microscopy, tomography or small-angle X-ray scattering are described. Situs is a modular program package for the multi-scale modeling of atomic resolution structures and low-resolution biophysical data from electron microscopy, tomography or small-angle X-ray scattering. This article provides an overview of recent developments in the Situs package, with an emphasis on workflows and conventions that are important for practical applications. The modular design of the programs facilitates scripting in the bash shell that allows specific programs tomore » be combined in creative ways that go beyond the original intent of the developers. Several scripting-enabled functionalities, such as flexible transformations of data type, the use of symmetry constraints or the creation of two-dimensional projection images, are described. The processing of low-resolution biophysical maps in such workflows follows not only first principles but often relies on implicit conventions. Situs conventions related to map formats, resolution, correlation functions and feature detection are reviewed and summarized. The compatibility of the Situs workflow with CCP4 conventions and programs is discussed.« less

An atomic scale study of surface termination and digital alloy growth in InGaAs/AlAsSb multi-quantum wells.

PubMed

Mauger, S J C; Bozkurt, M; Koenraad, P M; Zhao, Y; Folliot, H; Bertru, N

2016-07-20

An atomic scale study has been performed to understand the influence of the (As,Sb) shutter sequences during interface formation on the optical properties of InGaAs/AlAsSb quantum wells. Our cross-sectional scanning tunneling microscopy results show that the onset of the Sb profile is steep in the Sb-containing layers whereas an appreciable segregation of Sb in the subsequently grown Sb free layers is observed. The steep rise of the Sb profile is due to extra Sb that is supplied to the surface prior to the growth of the Sb-containing layers. No relation is found between the (As,Sb) termination conditions of the Sb-containing layers and the resulting Sb profiles in the capping layers. Correspondingly we see that the optical properties of these quantum wells are also nearly independent on the (As,Sb) shutter sequences at the interface. Digital alloy growth in comparison to conventional molecular beam epitaxy growth was also explored. X-ray results suggest that the structural properties of the quantum well structures grown by conventional molecular beam epitaxy techniques are slightly better than those formed by digital alloy growth. However photoluminescence studies indicate that the digital alloy samples give rise to a more intense and broader photoluminescence emission. Cross-sectional scanning tunneling microscopy measurements reveal that lateral composition modulations present in the digital alloys are responsible for the enhancement of the photoluminescence intensity and inhomogeneous broadening.

A Comparative Study Between Smartphone-Based Microscopy and Conventional Light Microscopy in 1021 Dermatopathology Specimens.

PubMed

Jahan-Tigh, Richard R; Chinn, Garrett M; Rapini, Ronald P

2016-01-01

The incorporation of high-resolution cameras into smartphones has allowed for a variety of medical applications including the use of lens attachments that provide telescopic, macroscopic, and dermatoscopic data, but the feasibility and performance characteristics of such a platform for use in dermatopathology have not been described. To determine the diagnostic performance of a smartphone microscope compared to traditional light microscopy in dermatopathology specimens. A simple smartphone microscope constructed with a 3-mm ball lens was used to prospectively evaluate 1021 consecutive dermatopathology cases in a blinded fashion. Referred, consecutive specimens from the community were evaluated at a single university hospital. The performance characteristics of the smartphone platform were calculated by using conventional light microscopy as the gold standard. The sensitivity and specificity for the diagnosis of melanoma, nonmelanoma skin cancers, and other miscellaneous conditions by the phone microscopy platform, as compared with traditional light microscopy, were calculated. For basal cell carcinoma (n = 136), the sensitivity and specificity of smartphone microscopy were 95.6% and 98.1%, respectively. The sensitivity and specificity for squamous cell carcinoma (n = 94) were 89.4% and 97.3%, respectively. The lowest sensitivity was found in melanoma (n = 15) at 60%, although the specificity was high at 99.1%. The accuracy of diagnosis of inflammatory conditions and other neoplasms was variable. Mobile phone-based microscopy has excellent performance characteristics for the inexpensive diagnosis of nonmelanoma skin cancers in a setting where a traditional microscope is not available.

Mechanical behavior of M-Wire and conventional NiTi wire used to manufacture rotary endodontic instruments.

PubMed

Pereira, Erika S J; Gomes, Renata O; Leroy, Agnès M F; Singh, Rupinderpal; Peters, Ove A; Bahia, Maria G A; Buono, Vicente T L

2013-12-01

Comparison of physical and mechanical properties of one conventional and a new NiTi wire, which had received an additional thermomechanical treatment. Specimens of both conventional (NiTi) and the new type of wire, called M-Wire (MW), were subjected to tensile and three-point bending tests, Vickers microhardness measurements, and to rotating-bending fatigue tests at a strain-controlled level of 6%. Fracture surfaces were observed by scanning electron microscopy and the non-deformed microstructures by transmission electron microscopy. The thermomechanical treatment applied to produce the M-Wire apparently increased the tensile strength and Vickers microhardness of the material, but its apparent Young modulus was smaller than that of conventionally treated NiTi. The three-point bending tests showed a higher flexibility for MW which also exhibited a significantly higher number of cycles to failure. M-Wire presented mechanical properties that can render endodontic instruments more flexible and fatigue resistant than those made with conventionally processed NiTi wires. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Functional cardiac magnetic resonance microscopy

NASA Astrophysics Data System (ADS)

Brau, Anja Christina Sophie

2003-07-01

The study of small animal models of human cardiovascular disease is critical to our understanding of the origin, progression, and treatment of this pervasive disease. Complete analysis of disease pathophysiology in these animal models requires measuring structural and functional changes at the level of the whole heart---a task for which an appropriate non-invasive imaging method is needed. The purpose of this work was thus to develop an imaging technique to support in vivo characterization of cardiac structure and function in rat and mouse models of cardiovascular disease. Whereas clinical cardiac magnetic resonance imaging (MRI) provides accurate assessment of the human heart, the extension of cardiac MRI from humans to rodents presents several formidable scaling challenges. Acquiring images of the mouse heart with organ definition and fluidity of contraction comparable to that achieved in humans requires an increase in spatial resolution by a factor of 3000 and an increase in temporal resolution by a factor of ten. No single technical innovation can meet the demanding imaging requirements imposed by the small animal. A functional cardiac magnetic resonance microscopy technique was developed by integrating improvements in physiological control, imaging hardware, biological synchronization of imaging, and pulse sequence design to achieve high-quality images of the murine heart with high spatial and temporal resolution. The specific methods and results from three different sets of imaging experiments are presented: (1) 2D functional imaging in the rat with spatial resolution of 175 mum2 x 1 mm and temporal resolution of 10 ms; (2) 3D functional imaging in the rat with spatial resolution of 100 mum 2 x 500 mum and temporal resolution of 30 ms; and (3) 2D functional imaging in the mouse with spatial resolution down to 100 mum2 x 1 mm and temporal resolution of 10 ms. The cardiac microscopy technique presented here represents a novel collection of technologies capable of acquiring routine high-quality images of murine cardiac structure and function with minimal artifacts and markedly higher spatial resolution compared to conventional techniques. This work is poised to serve a valuable role in the evaluation of cardiovascular disease and should find broad application in studies ranging from basic pathophysiology to drug discovery.

In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

NASA Astrophysics Data System (ADS)

Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

2018-02-01

A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

Time-Resolved Study of Nanomorphology and Nanomechanic Change of Early-Stage Mineralized Electrospun Poly(lactic acid) Fiber by Scanning Electron Microscopy, Raman Spectroscopy and Atomic Force Microscopy

PubMed Central

Wang, Mengmeng; Cai, Yin; Zhao, Bo; Zhu, Peizhi

2017-01-01

In this study, scanning electron microscopy (SEM), Raman spectroscopy and high-resolution atomic force microscopy (AFM) were used to reveal the early-stage change of nanomorphology and nanomechanical properties of poly(lactic acid) (PLA) fibers in a time-resolved manner during the mineralization process. Electrospun PLA nanofibers were soaked in simulated body fluid (SBF) for different periods of time (0, 1, 3, 5, 7 and 21 days) at 10 °C, much lower than the conventional 37 °C, to simulate the slow biomineralization process. Time-resolved Raman spectroscopy analysis can confirm that apatites were deposited on PLA nanofibers after 21 days of mineralization. However, there is no significant signal change among several Raman spectra before 21 days. SEM images can reveal the mineral deposit on PLA nanofibers during the process of mineralization. In this work, for the first time, time-resolved AFM was used to monitor early-stage nanomorphology and nanomechanical changes of PLA nanofibers. The Surface Roughness and Young’s Modulus of the PLA nanofiber quantitatively increased with the time of mineralization. The electrospun PLA nanofibers with delicate porous structure could mimic the extracellular matrix (ECM) and serve as a model to study the early-stage mineralization. Tested by the mode of PLA nanofibers, we demonstrated that AFM technique could be developed as a potential diagnostic tool to monitor the early onset of pathologic mineralization of soft tissues. PMID:28817096

Super-Resolution Microscopy of Cerebrospinal Fluid Biomarkers as a Tool for Alzheimer's Disease Diagnostics.

PubMed

Zhang, William I; Antonios, Gregory; Rabano, Alberto; Bayer, Thomas A; Schneider, Anja; Rizzoli, Silvio O

2015-01-01

Alzheimer's disease (AD) is neuropathologically characterized by aggregates of amyloid-β peptides (Aβ) and tau proteins. The consensus in the AD field is that Aβ and tau should serve as diagnostic biomarkers for AD. However, their aggregates have been difficult to investigate by conventional fluorescence microscopy, since their size is below the diffraction limit (∼200 nm). To solve this, we turned to a super-resolution imaging technique, stimulated emission depletion (STED) microscopy, which has a high enough precision to allow the discrimination of low- and high-molecular weight aggregates prepared in vitro. We used STED to analyze the structural organization of Aβ and tau in cerebrospinal fluid (CSF) from 36 AD patients, 11 patients with mild cognitive impairment (MCI), and 21 controls. We measured the numbers of aggregates in the CSF samples, and the aggregate sizes and intensities. These parameters enabled us to distinguish AD patients from controls with a specificity of ∼87% and a sensitivity of ∼79% . In addition, the aggregate parameters determined with STED microscopy correlated with the severity of cognitive impairment in AD patients. Finally, these parameters may be useful as predictive tools for MCI cases. The STED parameters of two MCI patients who developed AD during the course of the study, as well as of MCI patients whose Aβ ELISA values fall within the accepted range for AD, placed them close to the AD averages. We suggest that super-resolution imaging is a promising tool for AD diagnostics.

Investigation of sparsity metrics for autofocusing in digital holographic microscopy

NASA Astrophysics Data System (ADS)

Fan, Xin; Healy, John J.; Hennelly, Bryan M.

2017-05-01

Digital holographic microscopy (DHM) is an optoelectronic technique that is made up of two parts: (i) the recording of the interference pattern of the diffraction pattern of an object and a known reference wavefield using a digital camera and (ii) the numerical reconstruction of the complex object wavefield using the recorded interferogram and a distance parameter as input. The latter is based on the simulation of optical propagation from the camera plane to a plane at any arbitrary distance from the camera. A key advantage of DHM over conventional microscopy is that both the phase and intensity information of the object can be recovered at any distance, using only one capture, and this facilitates the recording of scenes that may change dynamically and that may otherwise go in and out of focus. Autofocusing using traditional microscopy requires mechanical movement of the translation stage or the microscope objective, and multiple image captures that are then compared using some metric. Autofocusing in DHM is similar, except that the sequence of intensity images, to which the metric is applied, is generated numerically from a single capture. We recently investigated the application of a number of sparsity metrics for DHM autofocusing and in this paper we extend this work to include more such metrics, and apply them over a greater range of biological diatom cells and magnification/numerical apertures. We demonstrate for the first time that these metrics may be grouped together according to matching behavior following high pass filtering.

Cell counting in whole mount tissue volumes using expansion OCT (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Yehe; Gu, Shi; Watanabe, Michiko; Rollins, Andrew M.; Jenkins, Michael W.

2017-02-01

Abnormal cell proliferation and migration during heart development can lead to severe congenital heart defects (CHDs). Studying the spatial distribution of cells during embryonic development helps our understanding of how the heart develops and the etiology of certain CHDs. However, imaging large groups of single cells in intact tissue volumes is challenging. No current technique can accomplish this task in both a time-efficient and cost-effective manner. OCT has potential with its large field of view and micron-scale resolution, but even the highest resolution OCT systems have poor contrast for counting cells and have a small field of view compared to conventional OCT. We propose using a conventional OCT system and processing the sample to enhance cellular contrast. Inspired by the recently developed Expansion Microscopy, we permeated whole-mount embryonic tissue with a superabsorbent monomer solution and polymerized into a hydrogel. When hydrated in DI water, the tissue-hydrogel complex was uniformly enlarged ( 5X in all dimensions) without distorting the microscopic structure. This had a twofold effect: it increased the resolution by a factor of 5 and decreased scattering, which allowed us to resolve cellular level features deep in the tissue with high contrast using conventional OCT. We noted that cell nuclei caused significantly more backscattering than the other subcellular structures after expansion. Based on this property, we were able to distinguish individual cell nuclei, and thus count cells, in expanded OCT images with simple intensity thresholding. We demonstrate the technique with embryonic quail hearts at various developmental stages.

In vivo microscopy of the mouse brain using multiphoton laser scanning techniques

NASA Astrophysics Data System (ADS)

Yoder, Elizabeth J.

2002-06-01

The use of multiphoton microscopy for imaging mouse brain in vivo offers several advantages and poses several challenges. This tutorial begins by briefly comparing multiphoton microscopy with other imaging modalities used to visualize the brain and its activity. Next, an overview of the techniques for introducing fluorescence into whole animals to generate contrast for in vivo microscopy using two-photon excitation is presented. Two different schemes of surgically preparing mice for brain imaging with multiphoton microscopy are reviewed. Then, several issues and problems with in vivo microscopy - including motion artifact, respiratory and cardiac rhythms, maintenance of animal health, anesthesia, and the use of fiducial markers - are discussed. Finally, examples of how these techniques have been applied to visualize the cerebral vasculature and its response to hypercapnic stimulation are provided.

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.

Comparison of marginal accuracy of castings fabricated by conventional casting technique and accelerated casting technique: an in vitro study.

PubMed

Reddy, S Srikanth; Revathi, Kakkirala; Reddy, S Kranthikumar

2013-01-01

Conventional casting technique is time consuming when compared to accelerated casting technique. In this study, marginal accuracy of castings fabricated using accelerated and conventional casting technique was compared. 20 wax patterns were fabricated and the marginal discrepancy between the die and patterns were measured using Optical stereomicroscope. Ten wax patterns were used for Conventional casting and the rest for Accelerated casting. A Nickel-Chromium alloy was used for the casting. The castings were measured for marginal discrepancies and compared. Castings fabricated using Conventional casting technique showed less vertical marginal discrepancy than the castings fabricated by Accelerated casting technique. The values were statistically highly significant. Conventional casting technique produced better marginal accuracy when compared to Accelerated casting. The vertical marginal discrepancy produced by the Accelerated casting technique was well within the maximum clinical tolerance limits. Accelerated casting technique can be used to save lab time to fabricate clinical crowns with acceptable vertical marginal discrepancy.

Xpert MTB/RIF assay for diagnosis of pulmonary tuberculosis in sputum specimens in remote health care facility.

PubMed

Geleta, Dereje Assefa; Megerssa, Yoseph Cherinet; Gudeta, Adugna Negussie; Akalu, Gizachew Taddesse; Debele, Melaku Tesfaye; Tulu, Kassu Desta

2015-10-19

Xpert MTB/RIF assay is considered as a great advance over conventional smear and culture in the diagnosis of TB and MDR-TB by simultaneously detecting M.tuberculosis and rifampicin resistance bacilli. However, very little information regarding the performance characteristics of Xpert MTB/RIF assay is available in Ethiopia. Therefore, the purpose of this study was to evaluate the performance of Xpert MTB/RIF assay compared to conventional sputum smear and culture methods for the diagnosis of pulmonary tuberculosis in remote health care facility. A paired expectorated sputum samples were obtained from 227 consecutively recruited patients with signs and symptoms suggestive of tuberculosis at Karamara hospital during December 2013 to May 2014. One of the sputum specimen was tested directly by Ziehl-Neelsen staining and Xpert MTB/RIF assay without NALC-NaOH decontamination. The other of pair of sputa specimen was cultured for isolation of TB bacilli by conventional methods. Diagnostic performance of Xpert MTB/RIF assay and AFB smear microscopy were calculated against culture as the gold standard. Overall 25.5% (58/227) samples were positive for Mycobacterium tuberculosis complex (MTBC) by MGIT and/or LJ media of which 36.2% (21/58) and 65.5% (35/58) were positive by AFB smear microscopy and Xpert MTB/RIF respectively. The sensitivity, specificity, as well as the positive and negative predictive value of Xpert MTB/RIF assay were 65.5% (95% CI: 53.3-77.7%), 96.3% (95% CI: 93.4-99.2%), 86.4% (95% CI: 76.2-96.5%), and 88.6% (95% CI: 83.9-93.3%) respectively. Eighteen of 58 (31%) cases that were smear microscopy negative, were positive by Xpert MTB/RIF assay. Although Xpert MTB/RIF assay demonstrated high sensitivity in detecting MTBC in sputum specimens compared with conventional AFB smear microscopy, it demonstrated suboptimal sensitivity in smear negative patients compared to conventional culture.

Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

PubMed

Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

1992-01-01

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.

Lightless cataract surgery using a near-infrared operating microscope.

PubMed

Kim, Bong-Hyun

2006-10-01

To describe the near-infrared (NIR) operating microscopy (NIOM) system using the NIR wavelength as the illumination source and to evaluate the feasibility of this system for lightless cataract surgery. HenAm Kim Eye Center, Haenam-Gun, South Korea. In this noncomparative interventional case series, cataract surgery was performed in 4 patients with bilateral cataract using the NIOM system in 1 eye and conventional microscopy in the fellow eye. The primary components of the system include an optical filter, a stereoscopic camera, head-mounted displays, and a recording system. This system uses invisible NIR (wavelength 850 to 1300 nm) illumination to facilitate cataract surgery without light. The differences between the NIOM system and conventional microscopy during cataract surgery were evaluated. The NIOM system provided excellent 3-dimensional viewing in real time. The image resolution was sufficient while performing all steps of cataract surgery. Immediately postoperatively and at 10 and 30 minutes and 1 hour, the visual acuity was better in the 4 eyes in which the NIOM system was used than in the 4 eyes in which conventional microscopy was used. However, using the NIOM system required good surgical skill. Lightless cataract surgery using the NIOM system seems useful for obtaining good visual acuity immediately postoperatively. The system may also reduce the incidence of light-induced retinal toxicity and the need for mydriatic administration and be a good educational tool.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Surface Characterization.

ERIC Educational Resources Information Center

Fulghum, J. E.; And Others

1989-01-01

This review is divided into the following analytical methods: ion spectroscopy, electron spectroscopy, scanning tunneling microscopy, atomic force microscopy, optical spectroscopy, desorption techniques, and X-ray techniques. (MVL)

Refixation of Osteochondral Fractures by an Ultrasound-Activated Pin System - An Ovine In Vivo Examination Using CT and Scanning Electron Microscope.

PubMed

H, Neumann; A P, Schulz; S, Breer; A, Unger; B, Kienast

2015-01-01

Osteochondral injuries, if not treated appropriately, often lead to severe osteoarthritis of the affected joint. Without refixation of the osteochondral fragment, human cartilage only repairs these defects imperfectly. All existing refixation systems for chondral defects have disadvantages, for instance bad MRI quality in the postoperative follow-up or low anchoring forces. To address the problem of reduced stability in resorbable implants, ultrasound-activated pins were developed. By ultrasound-activated melting of the tip of these implants a higher anchoring is assumed. Aim of the study was to investigate, if ultrasound-activated pins can provide a secure refixation of osteochondral fractures comparing to conventional screw and conventional, resorbable pin osteosynthesis. CT scans and scanning electron microscopy should proovegood refixation results with no further tissue damage by the melting of the ultrasound-activated pins in comparison to conventional osteosynthesis. Femoral osteochondral fragments in sheep were refixated with ultrasound-activated pins (SonicPin™), Ethipins(®) and screws (Asnis™). The quality of the refixated fragments was examined after three month of full weight bearing by CT scans and scanning electron microscopy of the cartilage surface. The CT examination found almost no statistically significant difference in the quality of refixation between the three different implants used. Concerning the CT morphology, ultrasound-activated pins demonstrated at least the same quality in refixation of osteochondral fragments as conventional resorbable pins or screws. The scanning electron microscopy showed no major surface damage by the three implants, especially any postulated cartilage damage induced by the heat of the ultrasound-activated pin. The screws protruded above the cartilage surface, which may affect the opposingtibial surface. Using CT scans and scanning electron microscopy, the SonicPin™, the Ethipin(®) and screws were at least equivalent in refixation quality of osteochondral fragments.

A computational approach to real-time image processing for serial time-encoded amplified microscopy

NASA Astrophysics Data System (ADS)

Oikawa, Minoru; Hiyama, Daisuke; Hirayama, Ryuji; Hasegawa, Satoki; Endo, Yutaka; Sugie, Takahisa; Tsumura, Norimichi; Kuroshima, Mai; Maki, Masanori; Okada, Genki; Lei, Cheng; Ozeki, Yasuyuki; Goda, Keisuke; Shimobaba, Tomoyoshi

2016-03-01

High-speed imaging is an indispensable technique, particularly for identifying or analyzing fast-moving objects. The serial time-encoded amplified microscopy (STEAM) technique was proposed to enable us to capture images with a frame rate 1,000 times faster than using conventional methods such as CCD (charge-coupled device) cameras. The application of this high-speed STEAM imaging technique to a real-time system, such as flow cytometry for a cell-sorting system, requires successively processing a large number of captured images with high throughput in real time. We are now developing a high-speed flow cytometer system including a STEAM camera. In this paper, we describe our approach to processing these large amounts of image data in real time. We use an analog-to-digital converter that has up to 7.0G samples/s and 8-bit resolution for capturing the output voltage signal that involves grayscale images from the STEAM camera. Therefore the direct data output from the STEAM camera generates 7.0G byte/s continuously. We provided a field-programmable gate array (FPGA) device as a digital signal pre-processor for image reconstruction and finding objects in a microfluidic channel with high data rates in real time. We also utilized graphics processing unit (GPU) devices for accelerating the calculation speed of identification of the reconstructed images. We built our prototype system, which including a STEAM camera, a FPGA device and a GPU device, and evaluated its performance in real-time identification of small particles (beads), as virtual biological cells, owing through a microfluidic channel.

Advances in photo-thermal infrared imaging microspectroscopy

NASA Astrophysics Data System (ADS)

Furstenberg, Robert; Kendziora, Chris; Papantonakis, Michael; Nguyen, Viet; McGill, Andrew

2013-05-01

There is a growing need for chemical imaging techniques in many fields of science and technology: forensics, materials science, pharmaceutical and chemical industries, just to name a few. While FTIR micro-spectroscopy is commonly used, its practical resolution limit of about 20 microns or more is often insufficient. Raman micro-spectroscopy provides better spatial resolution (~1 micron), but is not always practical because of samples exhibiting fluorescence or low Raman scattering efficiency. We are developing a non-contact and non-destructive technique we call photo-thermal infrared imaging spectroscopy (PT-IRIS). It involves photo-thermal heating of the sample with a tunable quantum cascade laser and measuring the resulting increase in thermal emission with an infrared detector. Photo-thermal emission spectra resemble FTIR absorbance spectra and can be acquired in both stand-off and microscopy configurations. Furthermore, PT-IRIS allows the acquisition of absorbance-like photo-thermal spectra in a reflected geometry, suitable for field applications and for in-situ study of samples on optically IR-opaque substrates (metals, fabrics, paint, glass etc.). Conventional FTIR microscopes in reflection mode measure the reflectance spectra which are different from absorbance spectra and are usually not catalogued in FTIR spectral libraries. In this paper, we continue developing this new technique. We perform a series of numerical simulations of the laser heating of samples during photo-thermal microscopy. We develop parameterized formulas to help the user pick the appropriate laser illumination power. We also examine the influence of sample geometry on spectral signatures. Finally, we measure and compare photo-thermal and reflectance spectra for two test samples.

Distortion of DNA Origami on Graphene Imaged with Advanced TEM Techniques.

PubMed

Kabiri, Yoones; Ananth, Adithya N; van der Torre, Jaco; Katan, Allard; Hong, Jin-Yong; Malladi, Sairam; Kong, Jing; Zandbergen, Henny; Dekker, Cees

2017-08-01

While graphene may appear to be the ultimate support membrane for transmission electron microscopy (TEM) imaging of DNA nanostructures, very little is known if it poses an advantage over conventional carbon supports in terms of resolution and contrast. Microscopic investigations are carried out on DNA origami nanoplates that are supported onto freestanding graphene, using advanced TEM techniques, including a new dark-field technique that is recently developed in our lab. TEM images of stained and unstained DNA origami are presented with high contrast on both graphene and amorphous carbon membranes. On graphene, the images of the origami plates show severe unwanted distortions, where the rectangular shape of the nanoplates is significantly distorted. From a number of comparative control experiments, it is demonstrated that neither staining agents, nor screening ions, nor the level of electron-beam irradiation cause this distortion. Instead, it is suggested that origami nanoplates are distorted due to hydrophobic interaction of the DNA bases with graphene upon adsorption of the DNA origami nanoplates. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eaton, Craig; Brahlek, Matthew; Engel-Herbert, Roman, E-mail: rue2@psu.edu

The authors report the growth of stoichiometric SrVO{sub 3} thin films on (LaAlO{sub 3}){sub 0.3}(Sr{sub 2}AlTaO{sub 6}){sub 0.7} (001) substrates using hybrid molecular beam epitaxy. This growth approach employs a conventional effusion cell to supply elemental A-site Sr and the metalorganic precursor vanadium oxytriisopropoxide (VTIP) to supply vanadium. Oxygen is supplied in its molecular form through a gas inlet. An optimal VTIP:Sr flux ratio has been identified using reflection high-energy electron-diffraction, x-ray diffraction, atomic force microscopy, and scanning transmission electron microscopy, demonstrating stoichiometric SrVO{sub 3} films with atomically flat surface morphology. Away from the optimal VTIP:Sr flux, characteristic changes inmore » the crystalline structure and surface morphology of the films were found, enabling identification of the type of nonstoichiometry. For optimal VTIP:Sr flux ratios, high quality SrVO{sub 3} thin films were obtained with smallest deviation of the lattice parameter from the ideal value and with atomically smooth surfaces, indicative of the good cation stoichiometry achieved by this growth technique.« less

The application of polyethylene glycol (PEG) to electron microscopy

PubMed Central

1980-01-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis. PMID:7400222

The application of polyethylene glycol (PEG) to electron microscopy.

PubMed

Wolosewick, J J

1980-08-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine-coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.

A hybrid analog-digital phase-locked loop for frequency mode non-contact scanning probe microscopy.

PubMed

Mehta, M M; Chandrasekhar, V

2014-01-01

Non-contact scanning probe microscopy (SPM) has developed into a powerful technique to image many different properties of samples. The conventional method involves monitoring the amplitude, phase, or frequency of a cantilever oscillating at or near its resonant frequency as it is scanned across the surface of a sample. For high Q factor cantilevers, monitoring the resonant frequency is the preferred method in order to obtain reasonable scan times. This can be done by using a phase-locked-loop (PLL). PLLs can be obtained as commercial integrated circuits, but these do not have the frequency resolution required for SPM. To increase the resolution, all-digital PLLs requiring sophisticated digital signal processors or field programmable gate arrays have also been implemented. We describe here a hybrid analog/digital PLL where most of the components are implemented using discrete analog integrated circuits, but the frequency resolution is provided by a direct digital synthesis chip controlled by a simple peripheral interface controller (PIC) microcontroller. The PLL has excellent frequency resolution and noise, and can be controlled and read by a computer via a universal serial bus connection.

A hybrid analog-digital phase-locked loop for frequency mode non-contact scanning probe microscopy

NASA Astrophysics Data System (ADS)

Mehta, M. M.; Chandrasekhar, V.

2014-01-01

Non-contact scanning probe microscopy (SPM) has developed into a powerful technique to image many different properties of samples. The conventional method involves monitoring the amplitude, phase, or frequency of a cantilever oscillating at or near its resonant frequency as it is scanned across the surface of a sample. For high Q factor cantilevers, monitoring the resonant frequency is the preferred method in order to obtain reasonable scan times. This can be done by using a phase-locked-loop (PLL). PLLs can be obtained as commercial integrated circuits, but these do not have the frequency resolution required for SPM. To increase the resolution, all-digital PLLs requiring sophisticated digital signal processors or field programmable gate arrays have also been implemented. We describe here a hybrid analog/digital PLL where most of the components are implemented using discrete analog integrated circuits, but the frequency resolution is provided by a direct digital synthesis chip controlled by a simple peripheral interface controller (PIC) microcontroller. The PLL has excellent frequency resolution and noise, and can be controlled and read by a computer via a universal serial bus connection.

Far-field optical imaging with subdiffraction resolution enabled by nonlinear saturation absorption

NASA Astrophysics Data System (ADS)

Ding, Chenliang; Wei, Jingsong

2016-01-01

The resolution of far-field optical imaging is required to improve beyond the Abbe limit to the subdiffraction or even the nanoscale. In this work, inspired by scanning electronic microscopy (SEM) imaging, in which carbon (or Au) thin films are usually required to be coated on the sample surface before imaging to remove the charging effect while imaging by electrons. We propose a saturation-absorption-induced far-field super-resolution optical imaging method (SAI-SRIM). In the SAI-SRIM, the carbon (or Au) layers in SEM imaging are replaced by nonlinear-saturation-absorption (NSA) thin films, which are directly coated onto the sample surfaces using advanced thin film deposition techniques. The surface fluctuant morphologies are replicated to the NSA thin films, accordingly. The coated sample surfaces are then imaged using conventional laser scanning microscopy. Consequently, the imaging resolution is greatly improved, and subdiffraction-resolved optical images are obtained theoretically and experimentally. The SAI-SRIM provides an effective and easy way to achieve far-field super-resolution optical imaging for sample surfaces with geometric fluctuant morphology characteristics.

Parametric Amplification Protocol for Frequency-Modulated Magnetic Resonance Force Microscopy Signals

NASA Astrophysics Data System (ADS)

Harrell, Lee; Moore, Eric; Lee, Sanggap; Hickman, Steven; Marohn, John

2011-03-01

We present data and theoretical signal and noise calculations for a protocol using parametric amplification to evade the inherent tradeoff between signal and detector frequency noise in force-gradient magnetic resonance force microscopy signals, which are manifested as a modulated frequency shift of a high- Q microcantilever. Substrate-induced frequency noise has a 1 / f frequency dependence, while detector noise exhibits an f2 dependence on modulation frequency f . Modulation of sample spins at a frequency that minimizes these two contributions typically results in a surface frequency noise power an order of magnitude or more above the thermal limit and may prove incompatible with sample spin relaxation times as well. We show that the frequency modulated force-gradient signal can be used to excite the fundamental resonant mode of the cantilever, resulting in an audio frequency amplitude signal that is readily detected with a low-noise fiber optic interferometer. This technique allows us to modulate the force-gradient signal at a sufficiently high frequency so that substrate-induced frequency noise is evaded without subjecting the signal to the normal f2 detector noise of conventional demodulation.

Transmission electron microscopy of unstained hybrid Au nanoparticles capped with PPAA (plasma-poly-allylamine): structure and electron irradiation effects.

PubMed

Gontard, Lionel C; Fernández, Asunción; Dunin-Borkowski, Rafal E; Kasama, Takeshi; Lozano-Pérez, Sergio; Lucas, Stéphane

2014-12-01

Hybrid (organic shell-inorganic core) nanoparticles have important applications in nanomedicine. Although the inorganic components of hybrid nanoparticles can be characterized readily using conventional transmission electron microscopy (TEM) techniques, the structural and chemical arrangement of the organic molecular components remains largely unknown. Here, we apply TEM to the physico-chemical characterization of Au nanoparticles that are coated with plasma-polymerized-allylamine, an organic compound with the formula C3H5NH2. We discuss the use of energy-filtered TEM in the low-energy-loss range as a contrast enhancement mechanism for imaging the organic shells of such particles. We also study electron-beam-induced crystallization and amorphization of the shells and the formation of graphitic-like layers that contain both C and N. The resistance of the samples to irradiation by high-energy electrons, which is relevant for optical tuning and for understanding the degree to which such hybrid nanostructures are stable in the presence of biomedical radiation, is also discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

A study of laser surface treatment in bonded repair of composite aircraft structures.

PubMed

Li, Shaolong; Sun, Ting; Liu, Chang; Yang, Wenfeng; Tang, Qingru

2018-03-01

Surface pre-treatment is one of the key processes in bonded repair of aircraft carbon fibre reinforced polymer composites. This paper investigates the surface modification of physical and chemical properties by laser ablation and conventional polish treatment techniques. Surface morphology analysed by laser scanning confocal microscopy and scanning electron microscopy showed that a laser-treated surface displayed higher roughness than that of a polish-treated specimen. The laser-treated laminate exhibited more functional groups in the form of O 1 s/C 1 s atomic ratio of 30.89% for laser-treated and 20.14% for polish-treated as evidenced by X-ray photoelectron spectroscopy observation. Contact angle goniometry demonstrated that laser treatment can provide increased surface free energy and wettability. In the light of mechanical interlocking, molecular bonding and thermodynamics theories on adhesion, laser etching process displayed enhanced bonding performance relative to the polishing surface treatment. These properties resulted in an increased single lap shear strength and a cohesive failure mode for laser etching while an adhesive failure mode occurred in polish-treated specimen.

Submicron mass spectrometry imaging of single cells by combined use of mega electron volt time-of-flight secondary ion mass spectrometry and scanning transmission ion microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siketić, Zdravko; Bogdanović Radović, Ivančica; Jakšić, Milko

In order to better understand biochemical processes inside an individual cell, it is important to measure the molecular composition at the submicron level. One of the promising mass spectrometry imaging techniques that may be used to accomplish this is Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS), using MeV energy heavy ions for excitation. MeV ions have the ability to desorb large intact molecules with a yield that is several orders of magnitude higher than conventional SIMS using keV ions. In order to increase the spatial resolution of the MeV TOF-SIMS system, we propose an independent TOF trigger using a STIM (scanningmore » transmission ion microscopy) detector that is placed just behind the thin transmission target. This arrangement is suitable for biological samples in which the STIM detector simultaneously measures the mass distribution in scanned samples. The capability of the MeV TOF-SIMS setup was demonstrated by imaging the chemical composition of CaCo-2 cells.« less

A study of laser surface treatment in bonded repair of composite aircraft structures

NASA Astrophysics Data System (ADS)

Li, Shaolong; Sun, Ting; Liu, Chang; Yang, Wenfeng; Tang, Qingru

2018-03-01

Surface pre-treatment is one of the key processes in bonded repair of aircraft carbon fibre reinforced polymer composites. This paper investigates the surface modification of physical and chemical properties by laser ablation and conventional polish treatment techniques. Surface morphology analysed by laser scanning confocal microscopy and scanning electron microscopy showed that a laser-treated surface displayed higher roughness than that of a polish-treated specimen. The laser-treated laminate exhibited more functional groups in the form of O 1 s/C 1 s atomic ratio of 30.89% for laser-treated and 20.14% for polish-treated as evidenced by X-ray photoelectron spectroscopy observation. Contact angle goniometry demonstrated that laser treatment can provide increased surface free energy and wettability. In the light of mechanical interlocking, molecular bonding and thermodynamics theories on adhesion, laser etching process displayed enhanced bonding performance relative to the polishing surface treatment. These properties resulted in an increased single lap shear strength and a cohesive failure mode for laser etching while an adhesive failure mode occurred in polish-treated specimen.

Confocal microscopy patterns in nonmelanoma skin cancer and clinical applications.

PubMed

González, S; Sánchez, V; González-Rodríguez, A; Parrado, C; Ullrich, M

2014-06-01

Reflectance confocal microscopy is currently the most promising noninvasive diagnostic tool for studying cutaneous structures between the stratum corneum and the superficial reticular dermis. This tool gives real-time images parallel to the skin surface; the microscopic resolution is similar to that of conventional histology. Numerous studies have identified the main confocal features of various inflammatory skin diseases and tumors, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings. Confocal patterns and diagnostic algorithms have been shown to have high sensitivity and specificity in melanoma and nonmelanoma skin cancer. Possible present and future applications of this noninvasive technology are wide ranging and reach beyond its use in noninvasive diagnosis. This tool can also be used, for example, to evaluate dynamic skin processes that occur after UV exposure or to assess tumor response to noninvasive treatments such as photodynamic therapy. We explain the characteristic confocal features found in the main nonmelanoma skin tumors and discuss possible applications for this novel diagnostic technique in routine dermatology practice. Copyright © 2012 Elsevier España, S.L. and AEDV. All rights reserved.

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging

PubMed Central

Bhattacharya, Dipanjan; Singh, Vijay Raj; Zhi, Chen; So, Peter T. C.; Matsudaira, Paul; Barbastathis, George

2012-01-01

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, “3D HiLo” where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts. PMID:23262684

Three dimensional HiLo-based structured illumination for a digital scanned laser sheet microscopy (DSLM) in thick tissue imaging.

PubMed

Bhattacharya, Dipanjan; Singh, Vijay Raj; Zhi, Chen; So, Peter T C; Matsudaira, Paul; Barbastathis, George

2012-12-03

Laser sheet based microscopy has become widely accepted as an effective active illumination method for real time three-dimensional (3D) imaging of biological tissue samples. The light sheet geometry, where the camera is oriented perpendicular to the sheet itself, provides an effective method of eliminating some of the scattered light and minimizing the sample exposure to radiation. However, residual background noise still remains, limiting the contrast and visibility of potentially interesting features in the samples. In this article, we investigate additional structuring of the illumination for improved background rejection, and propose a new technique, "3D HiLo" where we combine two HiLo images processed from orthogonal directions to improve the condition of the 3D reconstruction. We present a comparative study of conventional structured illumination based demodulation methods, namely 3Phase and HiLo with a newly implemented 3D HiLo approach and demonstrate that the latter yields superior signal-to-background ratio in both lateral and axial dimensions, while simultaneously suppressing image processing artifacts.

A study of laser surface treatment in bonded repair of composite aircraft structures

PubMed Central

Sun, Ting; Liu, Chang; Yang, Wenfeng; Tang, Qingru

2018-01-01

Surface pre-treatment is one of the key processes in bonded repair of aircraft carbon fibre reinforced polymer composites. This paper investigates the surface modification of physical and chemical properties by laser ablation and conventional polish treatment techniques. Surface morphology analysed by laser scanning confocal microscopy and scanning electron microscopy showed that a laser-treated surface displayed higher roughness than that of a polish-treated specimen. The laser-treated laminate exhibited more functional groups in the form of O 1 s/C 1 s atomic ratio of 30.89% for laser-treated and 20.14% for polish-treated as evidenced by X-ray photoelectron spectroscopy observation. Contact angle goniometry demonstrated that laser treatment can provide increased surface free energy and wettability. In the light of mechanical interlocking, molecular bonding and thermodynamics theories on adhesion, laser etching process displayed enhanced bonding performance relative to the polishing surface treatment. These properties resulted in an increased single lap shear strength and a cohesive failure mode for laser etching while an adhesive failure mode occurred in polish-treated specimen. PMID:29657748

Assessment of the microstructure evolution of an austempered ductile iron during austempering process through strain hardening analysis

NASA Astrophysics Data System (ADS)

Donnini, Riccardo; Fabrizi, Alberto; Bonollo, Franco; Zanardi, Franco; Angella, Giuliano

2017-09-01

The aim of this investigation was to determine a procedure based on tensile testing to assess the critical range of austempering times for having the best ausferrite produced through austempering. The austempered ductile iron (ADI) 1050 was quenched at different times during austempering and the quenched samples were tested in tension. The dislocation-density-related constitutive equation proposed by Estrin for materials having high density of geometrical obstacles to dislocation motion, was used to model the flow curves of the tensile tested samples. On the basis of strain hardening theory, the equation parameters were related to the microstructure of the quenched samples and were used to assess the ADI microstructure evolution during austempering. The microstructure evolution was also analysed through conventional optical microscopy, electron back-scattered diffraction technique and transmission electron microscopy. The microstructure observations resulted to be consistent with the assessment based on tensile testing, so the dislocation-density-related constitutive equation was found to be a powerful tool to characterise the evolution of the solid state transformations of austempering.

Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy.

PubMed

Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

2014-04-25

We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

Deconstructing Complexity: Serial Block-Face Electron Microscopic Analysis of the Hippocampal Mossy Fiber Synapse

PubMed Central

Wilke, Scott A.; Antonios, Joseph K.; Bushong, Eric A.; Badkoobehi, Ali; Malek, Elmar; Hwang, Minju; Terada, Masako; Ellisman, Mark H.

2013-01-01

The hippocampal mossy fiber (MF) terminal is among the largest and most complex synaptic structures in the brain. Our understanding of the development of this morphologically elaborate structure has been limited because of the inability of standard electron microscopy techniques to quickly and accurately reconstruct large volumes of neuropil. Here we use serial block-face electron microscopy (SBEM) to surmount these limitations and investigate the establishment of MF connectivity during mouse postnatal development. Based on volume reconstructions, we find that MF axons initially form bouton-like specializations directly onto dendritic shafts, that dendritic protrusions primarily arise independently of bouton contact sites, and that a dramatic increase in presynaptic and postsynaptic complexity follows the association of MF boutons with CA3 dendritic protrusions. We also identify a transient period of MF bouton filopodial exploration, followed by refinement of sites of synaptic connectivity. These observations enhance our understanding of the development of this highly specialized synapse and illustrate the power of SBEM to resolve details of developing microcircuits at a level not easily attainable with conventional approaches. PMID:23303931

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

PubMed Central

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

2010-01-01

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

sideSPIM - selective plane illumination based on a conventional inverted microscope.

PubMed

Hedde, Per Niklas; Malacrida, Leonel; Ahrar, Siavash; Siryaporn, Albert; Gratton, Enrico

2017-09-01

Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa . Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to 5 stacks/s. Additionally, sample handling is compatible with established methods and switching magnification to change the field of view from single cells to whole organisms does not require labor intensive adjustments of the system.

Fully automated three-dimensional microscopy system

NASA Astrophysics Data System (ADS)

Kerschmann, Russell L.

2000-04-01

Tissue-scale structures such as vessel networks are imaged at micron resolution with the Virtual Tissue System (VT System). VT System imaging of cubic millimeters of tissue and other material extends the capabilities of conventional volumetric techniques such as confocal microscopy, and allows for the first time the integrated 2D and 3D analysis of important tissue structural relationships. The VT System eliminates the need for glass slide-mounted tissue sections and instead captures images directly from the surface of a block containing a sample. Tissues are en bloc stained with fluorochrome compounds, embedded in an optically conditioned polymer that suppresses image signals form dep within the block , and serially sectioned for imaging. Thousands of fully registered 2D images are automatically captured digitally to completely convert tissue samples into blocks of high-resolution information. The resulting multi gigabyte data sets constitute the raw material for precision visualization and analysis. Cellular function may be seen in a larger anatomical context. VT System technology makes tissue metrics, accurate cell enumeration and cell cycle analyses possible while preserving full histologic setting.

IF-WS{sub 2} nanoparticles size design and synthesis via chemical reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghoreishi, S.M., E-mail: ghoreshi@cc.iut.ac.ir; Meshkat, S.S.; Dadkhah, A.A.

2010-05-15

An innovative synthesis of inorganic fullerene-like disulfide tungsten (IF-WS{sub 2}) nanoparticles was developed using a chemical reduction reaction in a horizontal quartz reactor. In this process, first tungsten trisulfide (WS{sub 3}) was formed via a chemical reaction of tetra thiotungstate ammonium ((NH{sub 4}){sub 2}WS{sub 4}), polyethylene glycol (PEG), and hydrochloric acid (HCl) at ambient temperature and pressure. Subsequently, WS{sub 3} was reacted with hydrogen (H{sub 2}) at high temperature (1173-1373 K) in a quartz tube. The produced WS{sub 2} nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray (EDAX), and transmission electron microscopy (TEM). Themore » characterization results indicated that the high-purity (100%) IF-WS{sub 2} nanoparticles were produced. Moreover, addition of surfactant (PEG) and higher operating temperature (1173-1373 K) decreased the particles agglomeration, and consequently led to the reduction of average diameter of WS{sub 2} particles in the range of 50-78 nm. The developed method is simple, environmentally compatible, and cost-effective in contrast to the conventional techniques.« less

Diffusive and inelastic scattering in ballistic-electron-emission spectroscopy and ballistic-electron-emission microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, E.Y.; Turner, B.R.; Schowalter, L.J.

1993-07-01

Ballistic-electron-emission microscopy (BEEM) of Au/Si(001) n type was done to study whether elastic scattering in the Au overlayer is dominant. It was found that there is no dependence of the BEEM current on the relative gradient of the Au surface with respect to the Si interface, and this demonstrates that significant elastic scattering must occur in the Au overlayer. Ballistic-electron-emission spectroscopy (BEES) was also done, and, rather than using the conventional direct-current BEES, alternating-current (ac) BEES was done on Au/Si and also on Au/PtSi/Si(001) n type. The technique of ac BEES was found to give linear threshold for the Schottkymore » barrier, and it also clearly showed the onset of electron-hole pair creation and other inelastic scattering events. The study of device quality PtSi in Au/PtSi/Si(001) yielded an attenuation length of 4 nm for electrons of energy 1 eV above the PtSi Fermi energy. 20 refs., 5 figs.« less

Micromechanical Characterization and Texture Analysis of Direct Cast Titanium Alloys Strips

NASA Technical Reports Server (NTRS)

2000-01-01

This research was conducted to determine a post-processing technique to optimize mechanical and material properties of a number of Titanium based alloys and aluminides processed via Melt Overflow Solidification Technique (MORST). This technique was developed by NASA for the development of thin sheet titanium and titanium aluminides used in high temperature applications. The materials investigated in this study included conventional titanium alloy strips and foils, Ti-1100, Ti-24Al-11Nb (Alpha-2), and Ti-48Al-2Ta (Gamma). The methodology used included micro-characterization, heat-treatment, mechanical processing and mechanical testing. Characterization techniques included optical, electron microscopy, and x-ray texture analysis. The processing included heat-treatment and mechanical deformation through cold rolling. The initial as-cast materials were evaluated for their microstructure and mechanical properties. Different heat-treatment and rolling steps were chosen to process these materials. The properties were evaluated further and a processing relationship was established in order to obtain an optimum processing condition. The results showed that the as-cast material exhibited a Widmanstatten (fine grain) microstructure that developed into a microstructure with larger grains through processing steps. The texture intensity showed little change for all processing performed in this investigation.

A New Technique for Measuring Concentration Dependence of Self and Collective Diffusivity by using a Single Sample

NASA Astrophysics Data System (ADS)

Sirorattanakul, Krittanon; Shen, Chong; Ou-Yang, Daniel

Diffusivity governs the dynamics of interacting particles suspended in a solvent. At high particle concentration, the interactions between particles become non-negligible, making the values of self and collective diffusivity diverge and concentration-dependent. Conventional methods for measuring this dependency, such as forced Rayleigh scattering, fluorescence correlation spectroscopy (FCS), and dynamic light scattering (DLS) require preparation of multiple samples. We present a new technique to measure this dependency by using only a single sample. Dielectrophoresis (DEP) is used to create concentration gradient in the solution. Across this concentration distribution, we use FCS to measure the concentration-dependent self diffusivity. Then, we switch off DEP to allow the particles to diffuse back to equilibrium. We obtain the time series of concentration distribution from fluorescence microscopy and use them to determine the concentration-dependent collective diffusivity. We compare the experimental results with computer simulations to verify the validity of this technique. Time and spatial resolution limits of FCS and imaging are also analyzed to estimate the limitation of the proposed technique. NSF DMR-0923299, Lehigh College of Arts and Sciences Undergraduate Research Grant, Lehigh Department of Physics, Emulsion Polymers Institute.

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

PubMed

Hickey, William J; Shetty, Ameesha R; Massey, Randall J; Toso, Daniel B; Austin, Jotham

2017-01-01

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Fabrication and characterization of bioactive glass-ceramic using soda-lime-silica waste glass.

PubMed

Abbasi, Mojtaba; Hashemi, Babak

2014-04-01

Soda-lime-silica waste glass was used to synthesize a bioactive glass-ceramic through solid-state reactions. In comparison with the conventional route, that is, the melt-quenching and subsequent heat treatment, the present work is an economical technique. Structural and thermal properties of the samples were examined by X-ray diffraction (XRD) and differential thermal analysis (DTA). The in vitro test was utilized to assess the bioactivity level of the samples by Hanks' solution as simulated body fluid (SBF). Bioactivity assessment by atomic absorption spectroscopy (AAS) and scanning electron microscopy (SEM) was revealed that the samples with smaller amount of crystalline phase had a higher level of bioactivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of conventional and new peritoneal dialysis fluids on leukocyte recruitment in the rat peritoneal membrane.

PubMed

Mortier, Siska; De Vriese, An S; McLoughlin, Rachel M; Topley, Nicholas; Schaub, Thomas P; Passlick-Deetjen, Jutta; Lameire, Norbert H

2003-05-01

Peritonitis remains an important cause of morbidity and technique failure in peritoneal dialysis (PD). Conventional peritoneal dialysate fluids (PDF) inhibit peritoneal leukocyte function in vitro and may thus adversely affect the immune response to peritonitis. New PDF have been designed with neutral pH, low glucose degradation product (GDP) contents, and bicarbonate as buffer. The present intravital microscopy study examined the effects of conventional and new PDF on leukocyte behavior in the peritoneal microcirculation of Wistar rats. The visceral peritoneum was superfused by a control solution (EBSS), a conventional (CAPD), or a new bicarbonate-buffered PDF with neutral pH and low GDP content (CAPD BicaVera). In addition, spent conventional and new PDF were tested. The number of rolling, adhering, and extravasated leukocytes and leukocyte rolling velocity were assessed at different time intervals after exposure to lipopolysaccharide (LPS) or cell-free supernatants of coagulase-negative staphylococci (CNS-CFS). Exposure to LPS or CNS-CFS dissolved in EBSS dramatically increased the number of rolling, adhering and extravasated leukocytes and decreased leukocyte rolling velocity. Superfusion by CAPD abolished the LPS- or CNS-CFS-induced leukocyte recruitment, whereas CAPD BicaVera had significantly fewer depressant effect. Spent PDF affected the leukocyte response in a similar way as fresh PDF. High lactate concentrations, GDP, and hypertonicity appeared to be mainly responsible for the inhibition of leukocyte recruitment. In conclusion, conventional PDF abolish in vivo leukocyte recruitment in response to potent inflammatory stimuli. Bicarbonate-buffered pH-neutral PDF with low GDP contents have fewer depressant effects and may therefore contribute to a better preservation of peritoneal host defense.

Attempt of correlative observation of morphological synaptic connectivity by combining confocal laser-scanning microscope and FIB-SEM for immunohistochemical staining technique.

PubMed

Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Honma, Satoru; Kaneko, Takeshi

2014-11-01

Ten years have passed since a serial block-face scanning electron microscopy (SBF-SEM) method was developed [1]. In this innovative method, samples were automatically sectioned with an ultramicrotome placed inside a scanning electron microscope column, and the block surfaces were imaged one after another by SEM to capture back-scattered electrons. The contrast-inverted images obtained by the SBF-SEM were very similar to those acquired using conventional TEM. SFB-SEM has made easy to acquire image stacks of the transmission electron microscopy (TEM) in the mesoscale, which is taken with the confocal laser-scanning microcopy(CF-LSM).Furthermore, serial-section SEM has been combined with the focused ion beam (FIB) milling method [2]. FIB-incorporated SEM (FIB-SEM) has enabled the acquisition of three-dimensional images with a higher z-axis resolution com- pared to ultramicrotome-equipped SEM.We tried immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in CF-LSM. Dendrites of neurons in the rat neostriatum were visualized using a recombinant viral vector. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively.We showed that conventional immuno-cytochemical staining for TEM was applicable to FIB-SEM. Furthermore, several synaptic contacts, which were thought to exist on the basis of CF-LSM findings, were confirmed with FIB-SEM, revealing the usefulness of the combined method of CF-LSM and FIB-SEM. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hyperlens-array-implemented optical microscopy

NASA Astrophysics Data System (ADS)

Iwanaga, Masanobu

2014-08-01

Limit of resolution of conventional optical microscopes has never reached below 100 nm under visible light illumination. We show that numerically designed high-transmittance hyperlens array (HLA) is implemented in an optical microscope and works in practice for achieving one-shot-recording optical images of in-situ placed objects with sub 50 nm resolution in lateral direction. Direct resolution test employing well-defined nanopatterns proves that the HLA-implemented imaging is super-resolution optical microscopy, which works even under nW/mm2 visible illumination for objects. The HLA implementation makes the resolution of conventional microscopes one-scale higher, leading to the 1/10 illumination wavelength range, that is, mesoscopic range.

[Iditification of five imported cases of Plasmodium ovale wallikeri infection in Zhejiang Province].

PubMed

Zhang, Ling-ling; Ruan, Wei; Chen, Hua-liang; Lu, Qiao-yi; Yao, Li-nong

2014-10-01

To identify and analyze Plasmodium ovale wallikeri in 5 imported malaria cases, who were detected positive by microscopy and negative by conventional PCR. Epidemiological information and blood samples were collected from the five patients. The detection was conducted by microscopy, Rapid Diagnostic Test (RDT) and nested PCR with Plasmodium genus-specific, species-specific and Plasmodium ovale wallikeri-specific primers. The amplified products were sequenced and Blast analysis was performed on line in NCBI. The five patients returned from Africa, and all had a history of malaria. They were microscopically positive for Plasmodium sp., and two cases showed Pan positive RDT result. All blood samples were negative for four Plasmodium spp. by conventional nested PCR, but positive by nested PCR with Plasmodium ovale wallikeri-specific primers. Blast analysis showed that the amplified sequences of the five cases had complete homology with P. ovale wallikeri clone RSH10 18S ribosomal RNA gene (Accession No. KF219561.1). The five cases which classified as positive by microscopy while negative by conventional PCR have been confirmed as Plasmodium ovale wallikeri infection by nested PCR with P. ovale wallikeri-specific primers.

Magnification concepts: The use of video-probe microscopy to stimulate excitement and hands-on discovery in the science classroom K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henk, C.; Garner, J.; Wandersee, J.H.

1994-12-31

We acquired and loaned several durable, easy-to use, though expensive video-probe microscopes. This hand-held, automatically focusing instrument can be used by a five year old and provides instant, excellent, in-focus images up to 200X on a video screen visible to all students simultaneously. The teacher is thus freed from the technical and logistic considerations involved in conventional classroom microscopy. K-12 teachers preview our videotape on probe utilization. They assemble and demonstrate the unit in the presence of our personnel, then check out the probe for use in their own classrooms. Extremely enthusiastic students examine samples ranging from their own fingerprintsmore » and clothing (on TV!) to pond water, prepared microscope slides, and microscope polarizing light phenomena. Teachers report heightened interest in conventional microscope use once the {open_quotes}microscopy connection{close_quotes} has been made.« less

Ultrastructural characterization of tooth-biomaterial interfaces prepared with broad and focused ion beams.

PubMed

Coutinho, E; Jarmar, T; Svahn, F; Neves, A A; Verlinden, B; Van Meerbeek, B; Engqvist, H

2009-11-01

Current available techniques for transmission electron microscopy (TEM) of tooth-biomaterial interfaces are mostly ineffective for brittle phases and impair integrated chemical and morphological characterization. The aims of this study were (1) to determine the applicability of new focused ion beam (FIB) and broad ion beam (BIB) techniques for TEM preparation of tooth-biomaterial interfaces; (2) to characterize the interfacial interaction with enamel and dentin of a conventional glass-ionomer (Chemfil Superior, DeTrey Dentsply, Germany), a 2-step self-etch (Clearfil SE, Kuraray, Japan) and a 3-step etch-and-rinse (OptiBond FL, Kerr, USA) adhesives; and (3) to characterize clinically relevant interfaces obtained from actual Class-I cavities. After bonding to freshly extracted human third molars, non-demineralized and non-stained sections were obtained using the FIB/BIB techniques and examined under TEM. The main structures generally disclosed in conventional ultramicrotomy samples were recognized in FIB/BIB-based ones. There were not any major differences between FIB and BIB concerning the resulting ultrastructural morphology. FIB/BIB-sections enabled to clearly resolve sub-micron hydroxyapatite crystals on top of hard tissues and the interface between matrix and filler in all materials, even at nano-scale. Some investigated interfaces disclosed areas with a distinct "fog" or "melted look", which is probably an artifact due to surface damage caused by the high-energy beam. Interfaces with enamel clearly disclosed the distinct "keyhole" shape of enamel rods sectioned at 90 degrees , delimited by a thin electron-lucent layer of inter-rod enamel. At regions where enamel crystals ran parallel with the interface, we observed a lack of interaction and some de-bonding along with interfacial void formation. The FIB/BIB methods are viable and reliable alternatives to conventional ultramicrotomy for preparation of thin sections of brittle and thus difficult to cut biomaterial-hard tissue interfaces. They disclose additional ultrastructural information about both substrates and are more suitable for advanced analytic procedures.

Marginal fit of all-ceramic crowns fabricated using two extraoral CAD/CAM systems in comparison with the conventional technique

PubMed Central

Alqahtani, Fawaz

2017-01-01

Objective The purpose of this study was to determine the effect of two extraoral computer-aided design (CAD) and computer-aided manufacturing (CAM) systems, in comparison with conventional techniques, on the marginal fit of monolithic CAD/CAM lithium disilicate ceramic crowns. Study design This is an in vitro interventional study. Place and duration of study The study was carried out at the Department of Prosthodontics, School of Dentistry, Prince Sattam Bin Abdul-Aziz University, Saudi Arabia, from December 2015 to April 2016. Methodology A marginal gap of 60 lithium disilicate crowns was evaluated by scanning electron microscopy. In total, 20 pressable lithium disilicate (IPS e.max Press [Ivoclar Vivadent]) ceramic crowns were fabricated using the conventional lost-wax technique as a control group. The experimental all-ceramic crowns were produced based on a scan stone model and milled using two extraoral CAD/CAM systems: the Cerec group was fabricated using the Cerec CAD/CAM system, and the Trios group was fabricated using Trios CAD and milled using Wieland Zenotec CAM. One-way analysis of variance (ANOVA) and the Scheffe post hoc test were used for statistical comparison of the groups (α=0.05). Results The mean (±standard deviation) of the marginal gap of each group was as follows: the Control group was 91.15 (±15.35) µm, the Cerec group was 111.07 (±6.33) µm, and the Trios group was 60.17 (±11.09) µm. One-way ANOVA and the Scheffe post hoc test showed a statistically significant difference in the marginal gap between all groups. Conclusion It can be concluded from the current study that all-ceramic crowns, fabricated using the CAD/CAM system, show a marginal accuracy that is acceptable in clinical environments. The Trios CAD group displayed the smallest marginal gap. PMID:28352204

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Superresolution Microscopy of the Nuclear Envelope and Associated Proteins.

PubMed

Xie, Wei; Horn, Henning F; Wright, Graham D

2016-01-01

Superresolution microscopy is undoubtedly one of the most exciting technologies since the invention of the optical microscope. Capable of nanometer-scale resolution to surpass the diffraction limit and coupled with the versatile labeling techniques available, it is revolutionizing the study of cell biology. Our understanding of the nucleus, the genetic and architectural center of the cell, has gained great advancements through the application of various superresolution microscopy techniques. This chapter describes detailed procedures of multichannel superresolution imaging of the mammalian nucleus, using structured illumination microscopy and single-molecule localization microscopy.

Mapping piezoelectric response in nanomaterials using a dedicated non-destructive scanning probe technique.

PubMed

Calahorra, Yonatan; Smith, Michael; Datta, Anuja; Benisty, Hadas; Kar-Narayan, Sohini

2017-12-14

There has been tremendous interest in piezoelectricity at the nanoscale, for example in nanowires and nanofibers where piezoelectric properties may be enhanced or controllably tuned, thus necessitating robust characterization techniques of piezoelectric response in nanomaterials. Piezo-response force microscopy (PFM) is a well-established scanning probe technique routinely used to image piezoelectric/ferroelectric domains in thin films, however, its applicability to nanoscale objects is limited due to the requirement for physical contact with an atomic force microscope (AFM) tip that may cause dislocation or damage, particularly to soft materials, during scanning. Here we report a non-destructive PFM (ND-PFM) technique wherein the tip is oscillated into "discontinuous" contact during scanning, while applying an AC bias between tip and sample and extracting the piezoelectric response for each contact point by monitoring the resulting localized deformation at the AC frequency. ND-PFM is successfully applied to soft polymeric (poly-l-lactic acid) nanowires, as well as hard ceramic (barium zirconate titanate-barium calcium titanate) nanowires, both previously inaccessible by conventional PFM. Our ND-PFM technique is versatile and compatible with commercial AFMs, and can be used to correlate piezoelectric properties of nanomaterials with their microstructural features thus overcoming key characterisation challenges in the field.

Chapter 14: Electron Microscopy on Thin Films for Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romero, Manuel; Abou-Ras, Daniel; Nichterwitz, Melanie

2016-07-22

This chapter overviews the various techniques applied in scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and highlights their possibilities and also limitations. It gives the various imaging and analysis techniques applied on a scanning electron microscope. The chapter shows that imaging is divided into that making use of secondary electrons (SEs) and of backscattered electrons (BSEs), resulting in different contrasts in the images and thus providing information on compositions, microstructures, and surface potentials. Whenever aiming for imaging and analyses at scales of down to the angstroms range, TEM and its related techniques are appropriate tools. In many cases,more » also SEM techniques provide the access to various material properties of the individual layers, not requiring specimen preparation as time consuming as TEM techniques. Finally, the chapter dedicates to cross-sectional specimen preparation for electron microscopy. The preparation decides indeed on the quality of imaging and analyses.« less

Microscopy basics and the study of actin-actin-binding protein interactions.

PubMed

Thomasson, Maggie S; Macnaughtan, Megan A

2013-12-15

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Digital Versus Conventional Impressions in Fixed Prosthodontics: A Review.

PubMed

Ahlholm, Pekka; Sipilä, Kirsi; Vallittu, Pekka; Jakonen, Minna; Kotiranta, Ulla

2018-01-01

To conduct a systematic review to evaluate the evidence of possible benefits and accuracy of digital impression techniques vs. conventional impression techniques. Reports of digital impression techniques versus conventional impression techniques were systematically searched for in the following databases: Cochrane Central Register of Controlled Trials, PubMed, and Web of Science. A combination of controlled vocabulary, free-text words, and well-defined inclusion and exclusion criteria guided the search. Digital impression accuracy is at the same level as conventional impression methods in fabrication of crowns and short fixed dental prostheses (FDPs). For fabrication of implant-supported crowns and FDPs, digital impression accuracy is clinically acceptable. In full-arch impressions, conventional impression methods resulted in better accuracy compared to digital impressions. Digital impression techniques are a clinically acceptable alternative to conventional impression methods in fabrication of crowns and short FDPs. For fabrication of implant-supported crowns and FDPs, digital impression systems also result in clinically acceptable fit. Digital impression techniques are faster and can shorten the operation time. Based on this study, the conventional impression technique is still recommended for full-arch impressions. © 2016 by the American College of Prosthodontists.

Marginal discrepancy dimensions of single unit metal crowns fabricated by using CAD-CAM-milled acrylate resin polymer blocks or a conventional waxing technique.

PubMed

Lalande, David; Hodd, Jeffrey A; Brousseau, John S; Ramos, Van; Dunham, Daniel; Rueggeberg, Frederick

2017-10-14

Because crowns with open margins are a well-known problem and can lead to complications, it is important to assess the accuracy of margins resulting from the use of a new technique. Currently, data regarding the marginal fit of computer-aided design and computer-aided manufacturing (CAD-CAM) technology to fabricate a complete gold crown (CGC) from a castable acrylate resin polymer block are lacking. The purpose of this in vitro study was to compare marginal discrepancy widths of CGCs fabricated by using either conventional hand waxing or acrylate resin polymer blocks generated by using CAD-CAM technology. A plastic model of a first mandibular molar was prepared by using a 1-mm, rounded chamfer margin on the entire circumference of the tooth. The master die was duplicated 30 times, and 15 wax patterns were fabricated by using a manual waxing technique, and 15 were fabricated by using CAD-CAM technology. All patterns were invested and cast, and resulting CGCs were cemented on their respective die by using resin-modified glass ionomer cement. The specimens were then embedded in acrylic resin and sectioned buccolingually. The buccal and lingual marginal discrepancies of each sectioned portion were measured by using microscopy at ×50 magnification. Data were subjected to repeated measures 2-way ANOVA, by using the Tukey post hoc pairwise comparison test (α=.05). The factor of "technique" had no significant influence on marginal discrepancy measurement (P=.431), but a significant effect of "margin location" (P=.019) was noted. The confounding combination of factors was found to be significantly lower marginal discrepancy dimensions of the lingual margin discrepancy than on the buccal side by using CAD-CAM technology. The marginal discrepancy of CAD-CAM acrylate resin crowns was not significantly different from those made with a conventional manual method; however, lingual margin discrepancies present from CAD-CAM-prepared crowns were significantly less than those measured on the respective buccal surface. Copyright © 2017 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Determination of Root Canal Cleanliness by Different Irrigation Methods and Morphometric Analysis of Apical Third.

PubMed

Seixas, Fábio Heredia; Estrela, Carlos; Bueno, Mike Reis; Sousa-Neto, Manoel Damião; Pécora, Jesus Djalma

2015-06-01

The aim of this study was to determine the root canal area before and after the instrumentation 1 mm short of the apical foramen by clinical and cone beam computed tomography (CBCT) methods, and to evaluate the cleanliness of the apical region in mesiodistal flattened teeth by using optical microscopy. Forty-two human single-canal mandibular incisors were instrumented using the Free Tip Preparation technique up to three, four or five instruments from the initial. Cone beam computed tomography scans were acquired of the samples before and after root canal preparation (RCP). Irrigation was performed by conventional or hydrodynamic means, using 2.5% sodium hypochlorite. The samples were prepared for observation under an optical microscope. Images were digitally obtained, analyzed and the results were submitted to statistical analysis (two-way ANOVA complemented by Bonferroni's post-test). There was no significant difference between the studied anatomical areas with both CBCT and clinical methods. There were no differences between irrigation methods. It was verified differences between instrumentation techniques. Instrumentation with four instruments from the initial instrument determined a significant increase in the contact area when compared to preparation with three instruments, but RCP with 5 instruments did not result in a better cleanliness. The analysis with CBCT was not capable to determine the precise shape of surgical apical area comparing to the clinical method. Both the conventional and hydrodynamic irrigation techniques were not able to promote root canals debris-free. The instruments action in root canal walls was proportional to the number of instruments used from the initial apical instrument.

Accuracy of Mobile Phone and Handheld Light Microscopy for the Diagnosis of Schistosomiasis and Intestinal Protozoa Infections in Côte d’Ivoire

PubMed Central

Coulibaly, Jean T.; Ouattara, Mamadou; D’Ambrosio, Michael V.; Fletcher, Daniel A.; Keiser, Jennifer; Utzinger, Jürg; N’Goran, Eliézer K.

2016-01-01

Background Handheld light microscopy using compact optics and mobile phones may improve the quality of health care in resource-constrained settings by enabling access to prompt and accurate diagnosis. Methodology Laboratory technicians were trained to operate two handheld diagnostic devices (Newton Nm1 microscope and a clip-on version of the mobile phone-based CellScope). The accuracy of these devices was compared to conventional light microscopy for the diagnosis of Schistosoma haematobium, S. mansoni, and intestinal protozoa infection in a community-based survey in rural Côte d’Ivoire. One slide of 10 ml filtered urine and a single Kato-Katz thick smear from 226 individuals were subjected to the Newton Nm1 microscope and CellScope for detection of Schistosoma eggs and compared to conventional microscopy. Additionally, 121 sodium acetate-acetic acid-formalin (SAF)-fixed stool samples were examined by the Newton Nm1 microscope and compared to conventional microscopy for the diagnosis of intestinal protozoa. Principal Findings The prevalence of S. haematobium, S. mansoni, Giardia intestinalis, and Entamoeba histolytica/E. dispar, as determined by conventional microscopy, was 39.8%, 5.3%, 20.7%, and 4.9%, respectively. The Newton Nm1 microscope had diagnostic sensitivities for S. mansoni and S. haematobium infection of 91.7% (95% confidence interval (CI) 59.8–99.6%) and 81.1% (95% CI 71.2–88.3%), respectively, and specificities of 99.5% (95% CI 97.0–100%) and 97.1% (95% CI 92.2–99.1%), respectively. The CellScope demonstrated sensitivities for S. mansoni and S. haematobium of 50.0% (95% CI 25.4–74.6%) and 35.6% (95% CI 25.9–46.4%), respectively, and specificities of 99.5% (95% CI 97.0–100%) and 100% (95% CI 86.7–100%), respectively. For G. intestinalis and E. histolytica/E. dispar, the Newton Nm1 microscope had sensitivity of 84.0% (95% CI 63.1–94.7%) and 83.3% (95% CI 36.5–99.1%), respectively, and 100% specificity. Conclusions/Significance Handheld diagnostic devices can be employed in community-based surveys in resource-constrained settings after minimal training of laboratory technicians to diagnose intestinal parasites. PMID:27348755

Experimental infection of conventional dogs with canine parvovirus.

PubMed

McAdaragh, J P; Eustis, S L; Nelson, D T; Stotz, I; Kenefick, K

1982-04-01

Four 6-week-old conventional pups were inoculated with a parvovirus (PV) isolated from the feces of a dog with naturally occurring enteritis. Blood for hematologic studies, virus isolation (VI), and antibody titration and feces for VI and negative-contrast electron microscopy were collected on day 0 and daily until necropsy. Beginning at postinoculation day 2, necropsies were done and specimens were collected for immunofluorescence, VI, and light microscopic examination. The PV infection was confirmed by VI, immunofluorescence, electron microscopy, and seroconversion. Clinical illness was not observed in inoculated pups, although mild intestinal lesions similar to those of naturally occurring PV enteritis were found. The failure to elicit severe disease in conventional pups indicates that one or more factors, such as intercurrent enteric or systemic infections, immune status, age, nutrition, virulence of virus, dose of infectious virus, and route of inoculation influence the clinical and pathologic manifestations of PV infection.

Transdermal delivery of raloxifene HCl via ethosomal system: Formulation, advanced characterizations and pharmacokinetic evaluation.

PubMed

Mahmood, Syed; Mandal, Uttam Kumar; Chatterjee, Bappaditya

2018-05-05

Raloxifene HCl belongs to a class of selective estrogen receptor modulators (SERMs) which is used for the management of breast cancer. The major problem reported with raloxifene is its poor bioavailability which is only up to 2%. The main objective of the present work was to formulate raloxifene loaded ethosomal preparation for transdermal application and compare it with an oral formulation of the drug. Five ethosomal formulations with different concentrations of ethanol and a conventional liposomes formulation were prepared by rotary evaporation method. The prepared systems were characterised by high resolution transmission electron microscopy (HRTEM), force emission electron microscopy (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and 31 P NMR study. All these advanced characterization study established that the ethosome formulation was well defined by its size, shape and its bilayer formation. Transdermal flux of the optimized ethosome formulation was 22.14 ± 0.83 µg/ml/cm 2 which was 21 times higher when compared to the conventional liposomes. Confocal microscopy study revealed an enhanced permeation of coumarin-6 dye loaded ethosomes to much deeper layers of skin when compared with conventional liposomes. The gel was found to be pseudoplastic with elastic behaviour. In-vivo studies on rats showed a higher bioavailability of RXL (157% times) for ethosomal formulation when compared with the oral formulation. In conclusion, RXL loaded ethosomal formulation via transdermal route showed superior drug delivery properties as compared to oral formulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Assessment of a Pan-Dermatophyte Nested-PCR Compared with Conventional Methods for Direct Detection and Identification of Dermatophytosis Agents in Animals.

PubMed

Piri, Fahimeh; Zarei Mahmoudabadi, Ali; Ronagh, Ali; Ahmadi, Bahram; Makimura, Koichi; Rezaei-Matehkolaei, Ali

2018-06-26

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Structure and short time degradation studies of sodium zirconium phosphate ceramics loaded with simulated fast breeder (FBR) waste

NASA Astrophysics Data System (ADS)

Ananthanarayanan, A.; Ambashta, R. D.; Sudarsan, V.; Ajithkumar, T.; Sen, D.; Mazumder, S.; Wattal, P. K.

2017-04-01

Sodium zirconium phosphate (NZP) ceramics have been prepared using conventional sintering and hot isostatic pressing (HIP) routes. The structure of NZP ceramics, prepared using the HIP route, has been compared with conventionally sintered NZP using a combination of X-ray diffraction (XRD) and (31P and 23Na) nuclear magnetic resonance (NMR) spectroscopy techniques. It is observed that NZP with no waste loading is aggressive toward the steel HIP-can during hot isostatic compaction and significant fraction of cations from the steel enter the ceramic material. Waste loaded NZP samples (10 wt% simulated FBR waste) show significantly low can-interaction and primary NZP phase is evident in this material. Upon exposure of can-interacted and waste loaded NZP to boiling water and steam, 31P NMR does not detect any major modifications in the network structure. However, the 23Na NMR spectra indicate migration of Na+ ions from the surface and possible re-crystallization. This is corroborated by Small-Angle Neutron Scattering (SANS) data and Scanning Electron Microscopy (SEM) measurements carried out on these samples.

Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy

PubMed Central

Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

2017-01-01

Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI’s conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components. PMID:28663887

Evaluation of marginal gap of Ni-Cr copings made with conventional and accelerated casting techniques.

PubMed

Tannamala, Pavan Kumar; Azhagarasan, Nagarasampatti Sivaprakasam; Shankar, K Chitra

2013-01-01

Conventional casting techniques following the manufacturers' recommendations are time consuming. Accelerated casting techniques have been reported, but their accuracy with base metal alloys has not been adequately studied. We measured the vertical marginal gap of nickel-chromium copings made by conventional and accelerated casting techniques and determined the clinical acceptability of the cast copings in this study. Experimental design, in vitro study, lab settings. Ten copings each were cast by conventional and accelerated casting techniques. All copings were identical, only their mold preparation schedules differed. Microscopic measurements were recorded at ×80 magnification on the perpendicular to the axial wall at four predetermined sites. The marginal gap values were evaluated by paired t test. The mean marginal gap by conventional technique (34.02 μm) is approximately 10 μm lesser than that of accelerated casting technique (44.62 μm). As the P value is less than 0.0001, there is highly significant difference between the two techniques with regard to vertical marginal gap. The accelerated casting technique is time saving and the marginal gap measured was within the clinically acceptable limits and could be an alternative to time-consuming conventional techniques.

Application of Ionic Liquids in the Microwave-Assisted Extraction of Proanthocyanidins from Larix gmelini Bark

PubMed Central

Yang, Lei; Sun, Xiaowei; Yang, Fengjian; Zhao, Chunjian; Zhang, Lin; Zu, Yuangang

2012-01-01

Ionic liquid based, microwave-assisted extraction (ILMAE) was successfully applied to the extraction of proanthocyanidins from Larix gmelini bark. In this work, in order to evaluate the performance of ionic liquids in the microwave-assisted extraction process, a series of 1-alkyl-3-methylimidazolium ionic liquids with different cations and anions were evaluated for extraction yield, and 1-butyl-3-methylimidazolium bromide was selected as the optimal solvent. In addition, the ILMAE procedure for the proanthocyanidins was optimized and compared with other conventional extraction techniques. Under the optimized conditions, satisfactory extraction yield of the proanthocyanidins was obtained. Relative to other methods, the proposed approach provided higher extraction yield and lower energy consumption. The Larix gmelini bark samples before and after extraction were analyzed by Thermal gravimetric analysis, Fourier-transform infrared spectroscopy and characterized by scanning electron microscopy. The results showed that the ILMAE method is a simple and efficient technique for sample preparation. PMID:22606036

Development of a Digital Microarray with Interferometric Reflectance Imaging

NASA Astrophysics Data System (ADS)

Sevenler, Derin

This dissertation describes a new type of molecular assay for nucleic acids and proteins. We call this technique a digital microarray since it is conceptually similar to conventional fluorescence microarrays, yet it performs enumerative ('digital') counting of the number captured molecules. Digital microarrays are approximately 10,000-fold more sensitive than fluorescence microarrays, yet maintain all of the strengths of the platform including low cost and high multiplexing (i.e., many different tests on the same sample simultaneously). Digital microarrays use gold nanorods to label the captured target molecules. Each gold nanorod on the array is individually detected based on its light scattering, with an interferometric microscopy technique called SP-IRIS. Our optimized high-throughput version of SP-IRIS is able to scan a typical array of 500 spots in less than 10 minutes. Digital DNA microarrays may have utility in applications where sequencing is prohibitively expensive or slow. As an example, we describe a digital microarray assay for gene expression markers of bacterial drug resistance.

Low-loss Ca{sub 5-x}Sr{sub x}A{sub 2}TiO{sub 12} [A=Nb,Ta] ceramics: Microwave dielectric properties and vibrational spectroscopic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bijumon, Pazhoor Varghese; Sebastian, Mailadil Thomas; Dias, Anderson

2005-05-15

Complex perovskite-type Ca{sub 5-x}Sr{sub x}A{sub 2}TiO{sub 12} [A=Nb,Ta] (0{<=}x{<=}5) ceramics were prepared by conventional solid-state ceramic route. The crystal structure, microwave dielectric properties, and vibrational spectroscopic characteristics of these materials are reported. The structure and microstructure were investigated by x-ray diffraction and scanning electron microscopy techniques. The microwave dielectric properties were measured in the 3-5-GHz frequency range by the resonance method. Structural evolutions from orthorhombic to an averaged pseudocubic phase, with associated changes in dielectric properties, were observed as a function of composition. The structure-property relationships in these ceramics were established using Raman and Fourier transform infrared spectroscopic techniques. Ramanmore » analysis showed characteristic bands of ordered perovskite materials, with variation in both intensity and frequency as a function of composition.« less

Spin-based diagnostic of nanostructure in copper phthalocyanine-C60 solar cell blends.

PubMed

Warner, Marc; Mauthoor, Soumaya; Felton, Solveig; Wu, Wei; Gardener, Jules A; Din, Salahud; Klose, Daniel; Morley, Gavin W; Stoneham, A Marshall; Fisher, Andrew J; Aeppli, Gabriel; Kay, Christopher W M; Heutz, Sandrine

2012-12-21

Nanostructure and molecular orientation play a crucial role in determining the functionality of organic thin films. In practical devices, such as organic solar cells consisting of donor-acceptor mixtures, crystallinity is poor and these qualities cannot be readily determined by conventional diffraction techniques, while common microscopy only reveals surface morphology. Using a simple nondestructive technique, namely, continuous-wave electron paramagnetic resonance spectroscopy, which exploits the well-understood angular dependence of the g-factor and hyperfine tensors, we show that in the solar cell blend of C(60) and copper phthalocyanine (CuPc)-for which X-ray diffraction gives no information-the CuPc, and by implication the C(60), molecules form nanoclusters, with the planes of the CuPc molecules oriented perpendicular to the film surface. This information demonstrates that the current nanostructure in CuPc:C(60) solar cells is far from optimal and suggests that their efficiency could be considerably increased by alternative film growth algorithms.

Potential-sensing electrochemical atomic force microscopy for in operando analysis of water-splitting catalysts and interfaces

NASA Astrophysics Data System (ADS)

Nellist, Michael R.; Laskowski, Forrest A. L.; Qiu, Jingjing; Hajibabaei, Hamed; Sivula, Kevin; Hamann, Thomas W.; Boettcher, Shannon W.

2018-01-01

Heterogeneous electrochemical phenomena, such as (photo)electrochemical water splitting to generate hydrogen using semiconductors and/or electrocatalysts, are driven by the accumulated charge carriers and thus the interfacial electrochemical potential gradients that promote charge transfer. However, measurements of the "surface" electrochemical potential during operation are not generally possible using conventional electrochemical techniques, which measure/control the potential of a conducting electrode substrate. Here we show that the nanoscale conducting tip of an atomic force microscope cantilever can sense the surface electrochemical potential of electrocatalysts in operando. To demonstrate utility, we measure the potential-dependent and thickness-dependent electronic properties of cobalt (oxy)hydroxide phosphate (CoPi). We then show that CoPi, when deposited on illuminated haematite (α-Fe2O3) photoelectrodes, acts as both a hole collector and an oxygen evolution catalyst. We demonstrate the versatility of the technique by comparing surface potentials of CoPi-decorated planar and mesoporous haematite and discuss viability for broader application in the study of electrochemical phenomena.

Rapid deposition of triangular silver nanoplates on planar surfaces: application to metal-enhanced fluorescence.

PubMed

Aslan, Kadir; Lakowicz, Joseph R; Geddes, Chris D

2005-04-07

A simple and rapid wet-chemical technique for the deposition of silver triangles on conventional glass substrates, which alleviates the need for lithography, has been developed. The technique is based on the seed-mediated cetyltrimethylammonium-bromide-directed growth of silver triangles on glass surfaces, where smaller spherical silver seeds that were attached to the surface were subsequently converted and grown into silver triangles in the presence of a cationic surfactant and silver ions. The size of the silver triangles was controlled by sequential immersion of silver seed-coated glass substrates into a growth solution and by the duration time of immersion. Atomic force microscopy studies revealed that the size of the silver triangles ranged between 100 and 500 nm. Interestingly, these new surfaces are a significant improvement over traditional silver island films for applications in metal-enhanced fluorescence. A routine 16-fold enhancement in emission intensity was typically observed, for protein-immobilized indocyanine green, with a relatively very low loading density of silver triangles on the glass surface.

Microchemical Analysis Of Space Operation Debris

NASA Technical Reports Server (NTRS)

Cummings, Virginia J.; Kim, Hae Soo

1995-01-01

Report discusses techniques used in analyzing debris relative to space shuttle operations. Debris collected from space shuttle, expendable launch vehicles, payloads carried by space shuttle, and payloads carried by expendable launch vehicles. Optical microscopy, scanning electron microscopy with energy-dispersive spectrometry, analytical electron microscopy with wavelength-dispersive spectrometry, and X-ray diffraction chosen as techniques used in examining samples of debris.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.