Large area scanning probe microscope in ultra-high vacuum demonstrated for electrostatic force measurements on high-voltage devices.

PubMed

Gysin, Urs; Glatzel, Thilo; Schmölzer, Thomas; Schöner, Adolf; Reshanov, Sergey; Bartolf, Holger; Meyer, Ernst

2015-01-01

The resolution in electrostatic force microscopy (EFM), a descendant of atomic force microscopy (AFM), has reached nanometre dimensions, necessary to investigate integrated circuits in modern electronic devices. However, the characterization of conducting or semiconducting power devices with EFM methods requires an accurate and reliable technique from the nanometre up to the micrometre scale. For high force sensitivity it is indispensable to operate the microscope under high to ultra-high vacuum (UHV) conditions to suppress viscous damping of the sensor. Furthermore, UHV environment allows for the analysis of clean surfaces under controlled environmental conditions. Because of these requirements we built a large area scanning probe microscope operating under UHV conditions at room temperature allowing to perform various electrical measurements, such as Kelvin probe force microscopy, scanning capacitance force microscopy, scanning spreading resistance microscopy, and also electrostatic force microscopy at higher harmonics. The instrument incorporates beside a standard beam deflection detection system a closed loop scanner with a scan range of 100 μm in lateral and 25 μm in vertical direction as well as an additional fibre optics. This enables the illumination of the tip-sample interface for optically excited measurements such as local surface photo voltage detection. We present Kelvin probe force microscopy (KPFM) measurements before and after sputtering of a copper alloy with chromium grains used as electrical contact surface in ultra-high power switches. In addition, we discuss KPFM measurements on cross sections of cleaved silicon carbide structures: a calibration layer sample and a power rectifier. To demonstrate the benefit of surface photo voltage measurements, we analysed the contact potential difference of a silicon carbide p/n-junction under illumination.

Importance of microscopy in durability studies of solidified and stabilized contaminated soils

USGS Publications Warehouse

Klich, I.; Wilding, L.P.; Drees, L.R.; Landa, E.R.

1999-01-01

Solidification/stabilization (S/S) is recognized by the U.S. EPA as a best demonstrated available technology for the containment of contaminated soils and other hazardous wastes that cannot be destroyed by chemical, thermal, or biological means. Despite the increased use of S/S technologies, little research has been conducted on the weathering and degradation of solidified and stabilized wastes once the treated materials have been buried. Published data to verify the performance and durability of landfilled treated wastes over time are rare. In this preliminary study, optical and electron microscopy (scanning electron microscopy [SEM], transmission electron microscopy [TEM] and electron probe microanalyses [EPMA]) were used to evaluate weathering features associated with metal-bearing contaminated soil that had been solidified and stabilized with Portland cement and subsequently buried on site, stored outdoors aboveground, or achieved in a laboratory warehouse for up to 6 yr. Physical and chemical alteration processes identified include: freeze-thaw cracking, cracking caused by the formation of expansive minerals such as ettringite, carbonation, and the movement of metals from waste aggregates into the cement micromass. Although the extent of degradation after 6 yr is considered slight to moderate, results of this study show that the same environmental concerns that affect the durability of concrete must be considered when evaluating the durability and permanence of the solidification and stabilization of contaminated soils with cement. In addition, such evaluations cannot be based on leaching and chemical analyses alone. The use of all levels of microscopic analyses must be incorporated into studies of the long-term performance of S/S technologies.Solidification/stabilization (S/S) is recognized by the U.S. EPA as a best demonstrated available technology for the containment of contaminated soils and other hazardous wastes that cannot be destroyed by chemical, thermal, or biological means. Despite the increased use of S/S technologies, little research has been conducted on the weathering and degradation of solidified and stabilized wastes once the treated materials have been buried. Published data to verify the performance and durability of landfilled treated wastes over time are rare. In this preliminary study, optical and electron microscopy (scanning electron microscopy [SEM], transmission electron microscopy [TEM] and electron probe microanalyses [EPMA]) were used to evaluate weathering features associated with metal-bearing contaminated soil that had been solidified and stabilized with Portland cement and subsequently buried on site, stored outdoors aboveground, or archived in a laboratory, warehouse for up to 6 yr. Physical and chemical alteration processes identified include: freeze-thaw cracking, cracking caused by the formation of expansive minerals such as ettringite, carbonation, and the movement of metals from waste aggregates into the cement micromass. Although the extent of degradation after 6 yr is considered slight to moderate, results of this study show that the same environmental concerns that affect the durability of concrete must be considered when evaluating the durability and permanence of the solidification and stabilization of contaminated soils with cement. In addition, such evaluations cannot be based on leaching and chemical analyses alone. The use of all levels of microscopic analyses must be incorporated into studies of the long-term performance of S/S technologies.

A rapid method for the assessment of bone architecture by confocal microscopy.

PubMed

Zheng, M H; Bruining, H G; Cody, S H; Brankov, B; Wood, D J; Papadimitriou, J M

1997-08-01

Conventional ways of demonstrating and analysing the components of osseous tissue have always been hampered by the difficulty of physically sectioning bone. In this study, we have used Acridine Orange staining of 100-micron-thick unembedded bone slices and then assessed the cellular and tissue architecture by confocal microscopy. The result showed the Acridine Orange, by differential staining of the cellular nucleic acids, permits ready assessment of cell shape and cell organization as well as variations in growth patterns. Our studies have provided a new and relatively easy way of assessing the morphology of bone specimens by rendering unnecessary the need for embedding, decalcification and thin sectioning of the osseous tissue.

Drinking water biofilms on copper and stainless steel exhibit specific molecular responses towards different disinfection regimes at waterworks.

PubMed

Jungfer, Christina; Friedrich, Frank; Varela Villarreal, Jessica; Brändle, Katharina; Gross, Hans-Jürgen; Obst, Ursula; Schwartz, Thomas

2013-09-01

Biofilms growing on copper and stainless steel substrata in natural drinking water were investigated. A modular pilot-scale distribution facility was installed at four waterworks using different raw waters and disinfection regimes. Three-month-old biofilms were analysed using molecular biology and microscopy methods. High total cell numbers, low counts of actively respiring cells and low numbers of cultivable bacteria indicated the high abundance of viable but not cultivable bacteria in the biofilms. The expression of the recA SOS responsive gene was detected and underlined the presence of transcriptionally active bacteria within the biofilms. This effect was most evident after UV disinfection, UV oxidation and UV disinfection with increased turbidity at waterworks compared to chemically treated and non-disinfected systems. Furthermore, live/dead staining techniques and environmental scanning electron microscopy imaging revealed the presence of living and intact bacteria in biofilms on copper substrata. Cluster analyses of DGGE profiles demonstrated differences in the composition of biofilms on copper and steel materials.

Ultrastructure and molecular phylogenetic position of a novel phagotrophic stramenopile from low oxygen environments: Rictus lutensis gen. et sp. nov. (Bicosoecida, incertae sedis).

PubMed

Yubuki, Naoji; Leander, Brian S; Silberman, Jeffrey D

2010-04-01

A novel free free-living phagotrophic flagellate, Rictus lutensis gen. et sp. nov., with two heterodynamic flagella, a permanent cytostome and a cytopharynx was isolated from muddy, low oxygen coastal sediments in Cape Cod, MA, USA. We cultivated and characterized this flagellate with transmission electron microscopy, scanning electron microscopy and molecular phylogenetic analyses inferred from small subunit (SSU) rDNA sequences. These data demonstrated that this organism has the key ultrastructural characters of the Bicosoecida, including similar transitional zones and a similar overall flagellar apparatus consisting of an x fiber and an L-shape microtubular root 2 involved in food capture. Although the molecular phylogenetic analyses were concordant with the ultrastructural data in placing R. lutensis with the bicosoecid clade, the internal position of this relatively divergent sequence within the clade was not resolved. Therefore, we interpret R. lutensis gen. et sp. nov. as a novel bicosoecid incertae sedis. Copyright 2009 Elsevier GmbH. All rights reserved.

Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

PubMed

Lin, Bo; Zhu, Mingyue; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

2017-10-01

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients. © 2017 UICC.

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

PubMed

Tran, Daniel N; Smith, Sandy A B C; Brown, David A; Parker, Andrew J C; Joseph, Joanne E; Armstrong, Nicola; Sewell, William A

2017-03-01

There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Synthesis of irregular graphene oxide tubes using green chemistry and their potential use as reinforcement materials for biomedical applications.

PubMed

Serrano-Aroca, Ángel; Deb, Sanjukta

2017-01-01

Micrometer length tubes of graphene oxide (GO) with irregular form were synthesised following facile and green metal complexation reactions. These materials were obtained by crosslinking of GO with calcium, zinc or strontium chlorides at three different temperatures (24, 34 and 55°C) using distilled water as solvent for the compounds and following a remarkably simple and low-cost synthetic method, which employs no hazardous substances and is conducted without consumption of thermal or sonic energy. These irregular continuous GO networks showed a very particular interconnected structure by Field Emission Scanning Electron Microscopy with Energy-Disperse X-Ray Spectroscopy for elemental analysis and High-resolution Transmission Electron Microscopy with Scanning Transmission Electron Microscope Dark Field Imaging, and were analysed by Raman Spectroscopy. To demonstrate the potential use of these 3D GO networks as reinforcement materials for biomedical applications, two composites of calcium alginate with irregular tubes of GO and with single GO nanosheets were prepared with the same amount of GO and divalent atoms and analysed. Thus, the dynamic-mechanical modulus of the composites synthesised with the 3D crosslinked GO networks showed a very significant mechanical improvement due to marked microstructural changes confirmed by confocal microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.

PubMed

Jaeger, Daniel; Pilger, Christian; Hachmeister, Henning; Oberländer, Elina; Wördenweber, Robin; Wichmann, Julian; Mussgnug, Jan H; Huser, Thomas; Kruse, Olaf

2016-10-21

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.

Synthesis of irregular graphene oxide tubes using green chemistry and their potential use as reinforcement materials for biomedical applications

PubMed Central

Deb, Sanjukta

2017-01-01

Micrometer length tubes of graphene oxide (GO) with irregular form were synthesised following facile and green metal complexation reactions. These materials were obtained by crosslinking of GO with calcium, zinc or strontium chlorides at three different temperatures (24, 34 and 55°C) using distilled water as solvent for the compounds and following a remarkably simple and low-cost synthetic method, which employs no hazardous substances and is conducted without consumption of thermal or sonic energy. These irregular continuous GO networks showed a very particular interconnected structure by Field Emission Scanning Electron Microscopy with Energy-Disperse X-Ray Spectroscopy for elemental analysis and High-resolution Transmission Electron Microscopy with Scanning Transmission Electron Microscope Dark Field Imaging, and were analysed by Raman Spectroscopy. To demonstrate the potential use of these 3D GO networks as reinforcement materials for biomedical applications, two composites of calcium alginate with irregular tubes of GO and with single GO nanosheets were prepared with the same amount of GO and divalent atoms and analysed. Thus, the dynamic-mechanical modulus of the composites synthesised with the 3D crosslinked GO networks showed a very significant mechanical improvement due to marked microstructural changes confirmed by confocal microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. PMID:28934354

Three-dimensional Organization of pKi-67: A Comparative Fluorescence and Electron Tomography Study Using Fluoronanogold

PubMed Central

Cheutin, Thierry; O'Donohue, Marie-Françoise; Beorchia, Adrien; Klein, Christophe; Kaplan, Hervé; Ploton, Dominique

2003-01-01

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250–300 nm in diameter, which are themselves composed of 30–50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin. PMID:14566014

Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using FluoroNanogold.

PubMed

Cheutin, Thierry; O'Donohue, Marie-Françoise; Beorchia, Adrien; Klein, Christophe; Kaplan, Hervé; Ploton, Dominique

2003-11-01

The monoclonal antibody (MAb) Ki-67 is routinely used in clinical studies to estimate the growth fraction of tumors. However, the role of pKi-67, the protein detected by the Ki-67 MAb, remains elusive, although some biochemical data strongly suggest that it might organize chromatin. To better understand the functional organization of pKi-67, we studied its three-dimensional distribution in interphase cells by confocal microscopy and electron tomography. FluoroNanogold, a single probe combining a dense marker with a fluorescent dye, was used to investigate pKi-67 organization at the optical and ultrastructural levels. Observation by confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli. Double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy studies confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250-300 nm in diameter, which are themselves composed of 30-50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.

Electric contributions to magnetic force microscopy response from graphene and MoS{sub 2} nanosheets

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

Li, Lu Hua, E-mail: luhua.li@deakin.edu.au; Chen, Ying

Magnetic force microscopy (MFM) signals have recently been detected from whole pieces of mechanically exfoliated graphene and molybdenum disulfide (MoS{sub 2}) nanosheets, and magnetism of the two nanomaterials was claimed based on these observations. However, non-magnetic interactions or artefacts are commonly associated with MFM signals, which make the interpretation of MFM signals not straightforward. A systematic investigation has been done to examine possible sources of the MFM signals from graphene and MoS{sub 2} nanosheets and whether the MFM signals can be correlated with magnetism. It is found that the MFM signals have significant non-magnetic contributions due to capacitive and electrostaticmore » interactions between the nanosheets and conductive cantilever tip, as demonstrated by electric force microscopy and scanning Kevin probe microscopy analyses. In addition, the MFM signals of graphene and MoS{sub 2} nanosheets are not responsive to reversed magnetic field of the magnetic cantilever tip. Therefore, the observed MFM response is mainly from electric artefacts and not compelling enough to correlate with magnetism of graphene and MoS{sub 2} nanosheets.« less

Paleomagnetic Analysis Using SQUID Microscopy

NASA Technical Reports Server (NTRS)

Weiss, Benjamin P.; Lima, Eduardo A.; Fong, Luis E.; Baudenbacher, Franz J.

2007-01-01

Superconducting quantum interference device (SQUID) microscopes are a new generation of instruments that map magnetic fields with unprecedented spatial resolution and moment sensitivity. Unlike standard rock magnetometers, SQUID microscopes map magnetic fields rather than measuring magnetic moments such that the sample magnetization pattern must be retrieved from source model fits to the measured field data. In this paper, we presented the first direct comparison between paleomagnetic analyses on natural samples using joint measurements from SQUID microscopy and moment magnetometry. We demonstrated that in combination with apriori geologic and petrographic data, SQUID microscopy can accurately characterize the magnetization of lunar glass spherules and Hawaiian basalt. The bulk moment magnitude and direction of these samples inferred from inversions of SQUID microscopy data match direct measurements on the same samples using moment magnetometry. In addition, these inversions provide unique constraints on the magnetization distribution within the sample. These measurements are among the most sensitive and highest resolution quantitative paleomagnetic studies of natural remanent magnetization to date. We expect that this technique will be able to extend many other standard paleomagnetic techniques to previously inaccessible microscale samples.

Conduction at domain walls in oxide multiferroics

NASA Astrophysics Data System (ADS)

Seidel, J.; Martin, L. W.; He, Q.; Zhan, Q.; Chu, Y.-H.; Rother, A.; Hawkridge, M. E.; Maksymovych, P.; Yu, P.; Gajek, M.; Balke, N.; Kalinin, S. V.; Gemming, S.; Wang, F.; Catalan, G.; Scott, J. F.; Spaldin, N. A.; Orenstein, J.; Ramesh, R.

2009-03-01

Domain walls may play an important role in future electronic devices, given their small size as well as the fact that their location can be controlled. Here, we report the observation of room-temperature electronic conductivity at ferroelectric domain walls in the insulating multiferroic BiFeO3. The origin and nature of the observed conductivity are probed using a combination of conductive atomic force microscopy, high-resolution transmission electron microscopy and first-principles density functional computations. Our analyses indicate that the conductivity correlates with structurally driven changes in both the electrostatic potential and the local electronic structure, which shows a decrease in the bandgap at the domain wall. Additionally, we demonstrate the potential for device applications of such conducting nanoscale features.

Conduction at domain walls in oxide multiferroics.

PubMed

Seidel, J; Martin, L W; He, Q; Zhan, Q; Chu, Y-H; Rother, A; Hawkridge, M E; Maksymovych, P; Yu, P; Gajek, M; Balke, N; Kalinin, S V; Gemming, S; Wang, F; Catalan, G; Scott, J F; Spaldin, N A; Orenstein, J; Ramesh, R

2009-03-01

Domain walls may play an important role in future electronic devices, given their small size as well as the fact that their location can be controlled. Here, we report the observation of room-temperature electronic conductivity at ferroelectric domain walls in the insulating multiferroic BiFeO(3). The origin and nature of the observed conductivity are probed using a combination of conductive atomic force microscopy, high-resolution transmission electron microscopy and first-principles density functional computations. Our analyses indicate that the conductivity correlates with structurally driven changes in both the electrostatic potential and the local electronic structure, which shows a decrease in the bandgap at the domain wall. Additionally, we demonstrate the potential for device applications of such conducting nanoscale features.

Synthesis of noble metal/carbon nanotube composites in supercritical methanol.

PubMed

Sun, Zhenyu; Fu, Lei; Liu, Zhimin; Han, Buxing; Liu, Yunqi; Du, Jimin

2006-03-01

A simple and efficient route has been employed to deposit noble metal nanoparticles (Pt, Ru, Pt-Ru, Rh, Ru-Sn) onto carbon nanotubes (CNTs) in supercritical methanol solution. In this method, the inorganic metallic salts acted as metal precursors, and methanol as solvent as well as reductant for the precursors. The as-prepared nanocomposites were structurally and morphologically characterized by X-ray diffraction spectroscopy, transmission electron microscopy (TEM), scanning electron microscopy, and X-ray photoelectron spectroscopy analyses. It was demonstrated that the CNTs were decorated by crystalline metal nanoparticles with uniform sizes and a narrow particle size distribution. The size and loading content of the nanoparticles on CNTs could be tuned by manipulating reaction parameters. Furthermore, the formation mechanism of the composites was also discussed.

Application of a real-space three-dimensional image reconstruction method in the structural analysis of noncrystalline biological macromolecules enveloped by water in coherent x-ray diffraction microscopy.

PubMed

Kodama, Wataru; Nakasako, Masayoshi

2011-08-01

Coherent x-ray diffraction microscopy is a novel technique in the structural analyses of particles that are difficult to crystallize, such as the biological particles composing living cells. As water is indispensable for maintaining particles in functional structures, sufficient hydration of targeted particles is required during sample preparation for diffraction microscopy experiments. However, the water enveloping particles also contributes significantly to the diffraction patterns and reduces the electron-density contrast of the sample particles. In this study, we propose a protocol for the structural analyses of particles in water by applying a three-dimensional reconstruction method in real space for the projection images phase-retrieved from diffraction patterns, together with a developed density modification technique. We examined the feasibility of the protocol through three simulations involving a protein molecule in a vacuum, and enveloped in either a droplet or a cube-shaped water. The simulations were carried out for the diffraction patterns in the reciprocal planes normal to the incident x-ray beam. This assumption and the simulation conditions corresponded to experiments using x-ray wavelengths of shorter than 0.03 Å. The analyses demonstrated that our protocol provided an interpretable electron-density map. Based on the results, we discuss the advantages and limitations of the proposed protocol and its practical application for experimental data. In particular, we examined the influence of Poisson noise in diffraction patterns on the reconstructed three-dimensional electron density in the proposed protocol.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

Attosecond electron pulse trains and quantum state reconstruction in ultrafast transmission electron microscopy

NASA Astrophysics Data System (ADS)

Priebe, Katharina E.; Rathje, Christopher; Yalunin, Sergey V.; Hohage, Thorsten; Feist, Armin; Schäfer, Sascha; Ropers, Claus

2017-12-01

Ultrafast electron and X-ray imaging and spectroscopy are the basis for an ongoing revolution in the understanding of dynamical atomic-scale processes in matter. The underlying technology relies heavily on laser science for the generation and characterization of ever shorter pulses. Recent findings suggest that ultrafast electron microscopy with attosecond-structured wavefunctions may be feasible. However, such future technologies call for means to both prepare and fully analyse the corresponding free-electron quantum states. Here, we introduce a framework for the preparation, coherent manipulation and characterization of free-electron quantum states, experimentally demonstrating attosecond electron pulse trains. Phase-locked optical fields coherently control the electron wavefunction along the beam direction. We establish a new variant of quantum state tomography—`SQUIRRELS'—for free-electron ensembles. The ability to tailor and quantitatively map electron quantum states will promote the nanoscale study of electron-matter entanglement and new forms of ultrafast electron microscopy down to the attosecond regime.

Fungal-Induced Deterioration of Mural Paintings: In Situ and Mock-Model Microscopy Analyses.

PubMed

Unković, Nikola; Grbić, Milica Ljaljević; Stupar, Miloš; Savković, Željko; Jelikić, Aleksa; Stanojević, Dragan; Vukojević, Jelena

2016-04-01

Fungal deterioration of frescoes was studied in situ on a selected Serbian church, and on a laboratory model, utilizing standard and newly implemented microscopy techniques. Scanning electron microscopy (SEM) with energy-dispersive X-ray confirmed the limestone components of the plaster. Pigments used were identified as carbon black, green earth, iron oxide, ocher, and an ocher/cinnabar mixture. In situ microscopy, applied via a portable microscope ShuttlePix P-400R, proved very useful for detection of invisible micro-impairments and hidden, symptomless, microbial growth. SEM and optical microscopy established that observed deterioration symptoms, predominantly discoloration and pulverization of painted layers, were due to bacterial filaments and fungal hyphal penetration, and formation of a wide range of fungal structures (i.e., melanized hyphae, chlamydospores, microcolonial clusters, Cladosporium-like conidia, and Chaetomium perithecia and ascospores). The all year-round monitoring of spontaneous and induced fungal colonization of a "mock painting" in controlled laboratory conditions confirmed the decisive role of humidity level (70.18±6.91% RH) in efficient colonization of painted surfaces, as well as demonstrated increased bioreceptivity of painted surfaces to fungal colonization when plant-based adhesives (ilinocopie, murdent), compared with organic adhesives of animal origin (bone glue, egg white), are used for pigment sizing.

MultiMap: A Tool to Automatically Extract and Analyse Spatial Microscopic Data From Large Stacks of Confocal Microscopy Images

PubMed Central

Varando, Gherardo; Benavides-Piccione, Ruth; Muñoz, Alberto; Kastanauskaite, Asta; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2018-01-01

The development of 3D visualization and reconstruction methods to analyse microscopic structures at different levels of resolutions is of great importance to define brain microorganization and connectivity. MultiMap is a new tool that allows the visualization, 3D segmentation and quantification of fluorescent structures selectively in the neuropil from large stacks of confocal microscopy images. The major contribution of this tool is the posibility to easily navigate and create regions of interest of any shape and size within a large brain area that will be automatically 3D segmented and quantified to determine the density of puncta in the neuropil. As a proof of concept, we focused on the analysis of glutamatergic and GABAergic presynaptic axon terminals in the mouse hippocampal region to demonstrate its use as a tool to provide putative excitatory and inhibitory synaptic maps. The segmentation and quantification method has been validated over expert labeled images of the mouse hippocampus and over two benchmark datasets, obtaining comparable results to the expert detections. PMID:29875639

MultiMap: A Tool to Automatically Extract and Analyse Spatial Microscopic Data From Large Stacks of Confocal Microscopy Images.

PubMed

Varando, Gherardo; Benavides-Piccione, Ruth; Muñoz, Alberto; Kastanauskaite, Asta; Bielza, Concha; Larrañaga, Pedro; DeFelipe, Javier

2018-01-01

The development of 3D visualization and reconstruction methods to analyse microscopic structures at different levels of resolutions is of great importance to define brain microorganization and connectivity. MultiMap is a new tool that allows the visualization, 3D segmentation and quantification of fluorescent structures selectively in the neuropil from large stacks of confocal microscopy images. The major contribution of this tool is the posibility to easily navigate and create regions of interest of any shape and size within a large brain area that will be automatically 3D segmented and quantified to determine the density of puncta in the neuropil. As a proof of concept, we focused on the analysis of glutamatergic and GABAergic presynaptic axon terminals in the mouse hippocampal region to demonstrate its use as a tool to provide putative excitatory and inhibitory synaptic maps. The segmentation and quantification method has been validated over expert labeled images of the mouse hippocampus and over two benchmark datasets, obtaining comparable results to the expert detections.

Electronegativity determination of individual surface atoms by atomic force microscopy.

PubMed

Onoda, Jo; Ondráček, Martin; Jelínek, Pavel; Sugimoto, Yoshiaki

2017-04-26

Electronegativity is a fundamental concept in chemistry. Despite its importance, the experimental determination has been limited only to ensemble-averaged techniques. Here, we report a methodology to evaluate the electronegativity of individual surface atoms by atomic force microscopy. By measuring bond energies on the surface atoms using different tips, we find characteristic linear relations between the bond energies of different chemical species. We show that the linear relation can be rationalized by Pauling's equation for polar covalent bonds. This opens the possibility to characterize the electronegativity of individual surface atoms. Moreover, we demonstrate that the method is sensitive to variation of the electronegativity of given atomic species on a surface due to different chemical environments. Our findings open up ways of analysing surface chemical reactivity at the atomic scale.

Electronegativity determination of individual surface atoms by atomic force microscopy

PubMed Central

Onoda, Jo; Ondráček, Martin; Jelínek, Pavel; Sugimoto, Yoshiaki

2017-01-01

Electronegativity is a fundamental concept in chemistry. Despite its importance, the experimental determination has been limited only to ensemble-averaged techniques. Here, we report a methodology to evaluate the electronegativity of individual surface atoms by atomic force microscopy. By measuring bond energies on the surface atoms using different tips, we find characteristic linear relations between the bond energies of different chemical species. We show that the linear relation can be rationalized by Pauling's equation for polar covalent bonds. This opens the possibility to characterize the electronegativity of individual surface atoms. Moreover, we demonstrate that the method is sensitive to variation of the electronegativity of given atomic species on a surface due to different chemical environments. Our findings open up ways of analysing surface chemical reactivity at the atomic scale. PMID:28443645

Magneto-optical Faraday rotation of semiconductor nanoparticles embedded in dielectric matrices.

PubMed

Savchuk, Andriy I; Stolyarchuk, Ihor D; Makoviy, Vitaliy V; Savchuk, Oleksandr A

2014-04-01

Faraday rotation has been studied for CdS, CdTe, and CdS:Mn semiconductor nanoparticles synthesized by colloidal chemistry methods. Additionally these materials were prepared in a form of semiconductor nanoparticles embedded in polyvinyl alcohol films. Transmission electron microscopy and atomic force microscopy analyses served as confirmation of nanocrystallinity and estimation of the average size of the nanoparticles. Spectral dependence of the Faraday rotation for the studied nanocrystals and nanocomposites is correlated with a blueshift of the absorption edge due to the confinement effect in zero-dimensional structures. Faraday rotation spectra and their temperature behavior in Mn-doped nanocrystals demonstrates peculiarities, which are associated with s, p-d exchange interaction between Mn²⁺ ions and band carriers in diluted magnetic semiconductor nanostructures.

Cloning and functional identification of moricins from the diamondback moth, Plutella xylostella (L.).

PubMed

Xia, X-F; Li, Y; Yu, X-Q; Lin, J-H; Li, S-Y; Li, Q; You, M-S

2017-10-01

Antimicrobial peptides (AMPs) are small-molecule peptides that play crucial roles in insect innate immune responses. To better understand the function of AMPs in Plutella xylostella, one of the main pests of cruciferous vegetables, three full-length cDNAs encoding moricins were cloned from Pl. xylostella. Two variants of the moricin named PxMor2 and PxMor3 were heterologously expressed and purified. A secondary structure analysis using circular dichroism demonstrated that the two peptides adopted an α-helical structure in the membrane-like environment, but in aqueous solution, they were present in random coiled conformation. Antimicrobial activity assays demonstrated that PxMor2 exhibited high activity against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli; however, PxMor3 only demonstrated high activity against E. coli. Scanning electron microscopy and confocal laser-scanning microscopy analyses suggest that PxMors can lead to the disruption of bacterial membrane, which might be the mechanism by which PxMors inhibit bacterial growth. This study contributes to the understanding of Pl. xylostella AMPs and immune responses, and also enriches the knowledge of insect moricin. © 2017 The Royal Entomological Society.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy

PubMed Central

Jaeger, Daniel; Pilger, Christian; Hachmeister, Henning; Oberländer, Elina; Wördenweber, Robin; Wichmann, Julian; Mussgnug, Jan H.; Huser, Thomas; Kruse, Olaf

2016-01-01

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation. PMID:27767024

Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes

PubMed Central

Baranovic, Jelena; Ramanujan, Chandra S.; Kasai, Nahoko; Midgett, Charles R.; Madden, Dean R.; Torimitsu, Keiichi; Ryan, John F.

2013-01-01

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. PMID:23382380

Formation of nanoparticles from thin silver films irradiated by laser pulses in air

NASA Astrophysics Data System (ADS)

Nastulyavichus, A. A.; Smirnov, N. A.; Kudryashov, S. I.; Ionin, A. A.; Saraeva, I. N.; Busleev, N. I.; Rudenko, A. A.; Khmel'nitskii, R. A.; Zayarnyi, D. A.

2018-03-01

Some specific features of the transport of silver nanoparticles onto a SiO2 substrate under focused nanosecond IR laser pulses is experimentally investigated. A possibility of obtaining silver coatings is demonstrated. The formation of silver nanostructures as a result of pulsed laser ablation in air is studied. Nanoparticles are formed by exposing a silver film to radiation of an HTF MARK (Bulat) laser marker (λ = 1064 nm). The thus prepared nanoparticles are analysed using scanning electron microscopy and optical spectroscopy.

Application of Membrane Crystallization for Minerals’ Recovery from Produced Water

PubMed Central

Ali, Aamer; Quist-Jensen, Cejna Anna; Macedonio, Francesca; Drioli, Enrico

2015-01-01

Produced water represents the largest wastewater stream from oil and gas production. Generally, its high salinity level restricts the treatment options. Membrane crystallization (MCr) is an emerging membrane process with the capability to extract simultaneously fresh water and valuable components from various streams. In the current study, the potential of MCr for produced water treatment and salt recovery was demonstrated. The experiments were carried out in lab scale and semi-pilot scale. The effect of thermal and hydrodynamic conditions on process performance and crystal characteristics were explored. Energy dispersive X-ray (EDX) and X-ray diffraction (XRD) analyses confirmed that the recovered crystals are sodium chloride with very high purity (>99.9%), also indicated by the cubic structure observed by microscopy and SEM (scanning electron microscopy) analysis. It was demonstrated experimentally that at recovery factor of 37%, 16.4 kg NaCl per cubic meter of produced water can be recovered. Anti-scaling surface morphological features of membranes were also identified. In general, the study provides a new perspective of isolation of valuable constituents from produced water that, otherwise, is considered as a nuisance. PMID:26610581

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Preparing the future post-mortem analysis of beryllium-based JET and ITER samples by multi-wavelengths Raman spectroscopy on implanted Be, and co-deposited Be

NASA Astrophysics Data System (ADS)

Rusu, M. I.; Pardanaud, C.; Ferro, Y.; Giacometti, G.; Martin, C.; Addab, Y.; Roubin, P.; Minissale, M.; Ferri, L.; Virot, F.; Barrachin, M.; Lungu, C. P.; Porosnicu, C.; Dinca, P.; Lungu, M.; Köppen, M.; Hansen, P.; Linsmeier, Ch.

2017-07-01

This study demonstrates that Raman microscopy is a suitable technique for future post mortem analyses of JET and ITER plasma facing components. We focus here on laboratory deposited and bombarded samples of beryllium and beryllium carbides and start to build a reference spectral databases for fusion relevant beryllium-based materials. We identified the beryllium phonon density of states, its second harmonic and E 2G and B 2G second harmonic and combination modes for defective beryllium in the spectral range 300-700 and 700-1300 cm-1, lying close to Be-D modes of beryllium hydrides. We also identified beryllium carbide signature, Be2C, combining Raman microscopy and DFT calculation. We have shown that, depending on the optical constants of the material probed, in depth sensitivity at the nanometer scale can be performed using different wavelengths. This way, we demonstrate that multi-wavelength Raman microscopy is sensitive to in-depth stress caused by ion implantation (down to  ≈30 nm under the surface for Be) and Be/C concentration (down to 400 nm or more under the surface for Be+C), which is a main contribution of this work. The depth resolution reached can then be adapted for studying the supersaturated surface layer found on tokamak deposits.

Automated fluorescent miscroscopic image analysis of PTBP1 expression in glioma

PubMed Central

Becker, Aline; Elder, Brad; Puduvalli, Vinay; Winter, Jessica; Gurcan, Metin

2017-01-01

Multiplexed immunofluorescent testing has not entered into diagnostic neuropathology due to the presence of several technical barriers, amongst which includes autofluorescence. This study presents the implementation of a methodology capable of overcoming the visual challenges of fluorescent microscopy for diagnostic neuropathology by using automated digital image analysis, with long term goal of providing unbiased quantitative analyses of multiplexed biomarkers for solid tissue neuropathology. In this study, we validated PTBP1, a putative biomarker for glioma, and tested the extent to which immunofluorescent microscopy combined with automated and unbiased image analysis would permit the utility of PTBP1 as a biomarker to distinguish diagnostically challenging surgical biopsies. As a paradigm, we utilized second resections from patients diagnosed either with reactive brain changes (pseudoprogression) and recurrent glioblastoma (true progression). Our image analysis workflow was capable of removing background autofluorescence and permitted quantification of DAPI-PTBP1 positive cells. PTBP1-positive nuclei, and the mean intensity value of PTBP1 signal in cells. Traditional pathological interpretation was unable to distinguish between groups due to unacceptably high discordance rates amongst expert neuropathologists. Our data demonstrated that recurrent glioblastoma showed more DAPI-PTBP1 positive cells and a higher mean intensity value of PTBP1 signal compared to resections from second surgeries that showed only reactive gliosis. Our work demonstrates the potential of utilizing automated image analysis to overcome the challenges of implementing fluorescent microscopy in diagnostic neuropathology. PMID:28282372

Translational Immunoimaging and Neuroimaging Demonstrate Corneal Neuroimmune Crosstalk.

PubMed

Hamrah, Pedram; Seyed-Razavi, Yashar; Yamaguchi, Takefumi

2016-11-01

Corneal immunoimaging and neuroimaging approaches facilitate in vivo analyses of the cornea, including high-resolution imaging of corneal immune cells and nerves. This approach facilitates the analyses of underlying immune and nerve alterations not detected by clinical slit-lamp examination alone. In this review, we describe recent work performed in our translational ocular immunology center with a focus on "bench-to-bedside" and "bedside-to-bench" research. The ability to visualize dendritiform immune cells (DCs) in patients with laser in vivo confocal microscopy (IVCM), recently discovered in the central murine cornea, has allowed us to demonstrate their utility as a potential surrogate biomarker for inflammatory ocular surface diseases. This biomarker for inflammation allows the measurement of therapeutic efficacy of anti-inflammatory drugs and its utility as an endpoint in clinical trials with high interobserver agreement. IVCM image analyses from our studies has demonstrated a significant increase in DC density and size in ocular disease, a positive correlation between DC density and clinical signs and symptoms of disease and pro-inflammatory tear cytokines, and a strong negative correlation between DC density and subbasal nerve density. In conjunction with preclinical research investigating the inflammatory state in a partial or fully denervated cornea, our results indicated that corneal nerves are directly involved in the regulation of homeostasis and immune privilege in the cornea.

Charge transport and intrinsic fluorescence in amyloid-like fibrils

PubMed Central

del Mercato, Loretta Laureana; Pompa, Pier Paolo; Maruccio, Giuseppe; Torre, Antonio Della; Sabella, Stefania; Tamburro, Antonio Mario; Cingolani, Roberto; Rinaldi, Ross

2007-01-01

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications. PMID:17984067

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

PubMed Central

Malide, Daniela; Métais, Jean-Yves; Dunbar, Cynthia E.

2014-01-01

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. PMID:25145579

Keratitis-associated fungi form biofilms with reduced antifungal drug susceptibility.

PubMed

Zhang, Xiaoyan; Sun, Xuguang; Wang, Zhiqun; Zhang, Yang; Hou, Wenbo

2012-11-21

To investigate the biofilm-forming capacity of Fusarium solani, Cladosporium sphaerospermum, and Acremonium implicatum, and the activities of antifungal agents against the three keratitis-associated fungi. The architecture of biofilms was analyzed using scanning electron microscopy and confocal scanning laser microscopy (CSLM). Susceptibility against six antifungal drugs was measured using the CLSI M38-A method and XTT reduction assay. Time course analyses of CSLM revealed that biofilm formation occurred in an organized fashion through four distinct developmental phases: adhesion, germling formation, microcolony formation, and biofilm maturation. Scanning electron microscopy revealed that mature biofilms displayed a complex three-dimensional structure, consisting of coordinated network of hyphal structures glued by the extracellular matrix (ECM). The antifungal susceptibility testing demonstrated a time-dependent decrease in efficacy for all six antifungal agents as the complexity of fungal hyphal structures developed. Natamycin (NAT), amphotericin B (AMB), and NAT were the most effective against F. solani, C. sphaerospermum, and A. implicatum biofilm, respectively. Corneal isolates of F. solani, C. sphaerospermum, and A. implicatum could produce biofilms that were resistant to antifungal agents in vitro.

Detection of bio-signature by microscopy and mass spectrometry

NASA Astrophysics Data System (ADS)

Tulej, M.; Wiesendanger, R.; Neuland, M., B.; Meyer, S.; Wurz, P.; Neubeck, A.; Ivarsson, M.; Riedo, V.; Moreno-Garcia, P.; Riedo, A.; Knopp, G.

2017-09-01

We demonstrate detection of micro-sized fossilized bacteria by means of microscopy and mass spectrometry. The characteristic structures of lifelike forms are visualized with a micrometre spatial resolution and mass spectrometric analyses deliver elemental and isotope composition of host and fossilized materials. Our studies show that high selectivity in isolation of fossilized material from host phase can be achieved while applying a microscope visualization (location), a laser ablation ion source with sufficiently small laser spot size and applying depth profiling method. Our investigations shows that fossilized features can be well isolated from host phase. The mass spectrometric measurements can be conducted with sufficiently high accuracy and precision yielding quantitative elemental and isotope composition of micro-sized objects. The current performance of the instrument allows the measurement of the isotope fractionation in per mill level and yield exclusively definition of the origin of the investigated species by combining optical visualization of investigated samples (morphology and texture), chemical characterization of host and embedded in the host micro-sized structure. Our isotope analyses involved bio-relevant B, C, S, and Ni isotopes which could be measured with sufficiently accuracy to conclude about the nature of the micro-sized objects.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

PubMed Central

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

2017-01-01

Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

Tracing cell lineages in videos of lens-free microscopy.

PubMed

Rempfler, Markus; Stierle, Valentin; Ditzel, Konstantin; Kumar, Sanjeev; Paulitschke, Philipp; Andres, Bjoern; Menze, Bjoern H

2018-06-05

In vitro experiments with cultured cells are essential for studying their growth and migration pattern and thus, for gaining a better understanding of cancer progression and its treatment. Recent progress in lens-free microscopy (LFM) has rendered it an inexpensive tool for label-free, continuous live cell imaging, yet there is only little work on analysing such time-lapse image sequences. We propose (1) a cell detector for LFM images based on fully convolutional networks and residual learning, and (2) a probabilistic model based on moral lineage tracing that explicitly handles multiple detections and temporal successor hypotheses by clustering and tracking simultaneously. (3) We benchmark our method in terms of detection and tracking scores on a dataset of three annotated sequences of several hours of LFM, where we demonstrate our method to produce high quality lineages. (4) We evaluate its performance on a somewhat more challenging problem: estimating cell lineages from the LFM sequence as would be possible from a corresponding fluorescence microscopy sequence. We present experiments on 16 LFM sequences for which we acquired fluorescence microscopy in parallel and generated annotations from them. Finally, (5) we showcase our methods effectiveness for quantifying cell dynamics in an experiment with skin cancer cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultrastructural characterization of the new NG97ht human-derived glioma cell line using two different electron microscopy technical procedures.

PubMed

Machado, Camila Maria Longo; Zorzeto, Tatiane Queiroz; Bianco, Juares E Romero; Rosa, Renata Giardini; Genari, Selma Candelaria; Joazeiro, Paulo Pinto; Verinaud, Liana

2009-04-01

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first.

Chemical analyses of fossil bone.

PubMed

Zheng, Wenxia; Schweitzer, Mary Higby

2012-01-01

The preservation of microstructures consistent with soft tissues, cells, and other biological components in demineralized fragments of dinosaur bone tens of millions of years old was unexpected, and counter to current hypotheses of tissue, cellular, and molecular degradation. Although the morphological similarity of these tissues to extant counterparts was unmistakable, after at least 80 million years exposed to geochemical influences, morphological similarity is insufficient to support an endogenous source. To test this hypothesis, and to characterize these materials at a molecular level, we applied multiple independent chemical, molecular, and microscopic analyses to identify the presence of original components produced by the extinct organisms. Microscopic techniques included field emission scanning electron microscopy, analytical transmission electron microscopy, transmitted light microscopy (LM), and fluorescence microscopy (FM). The chemical and molecular techniques include enzyme-linked immunosorbant assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot (immunoblot), and attenuated total reflectance infrared spectroscopy. In situ analyses performed directly on tissues included immunohistochemistry and time-of-flight secondary ion mass spectrometry. The details of sample preparation and methodology are described in detail herein.

Promising features of low-temperature grown Ge nanostructures on Si(001) substrates

NASA Astrophysics Data System (ADS)

Wang, Ze; Wang, Shuguang; Yin, Yefei; Liu, Tao; Lin, Dongdong; Li, De-hui; Yang, Xinju; Jiang, Zuimin; Zhong, Zhenyang

2017-03-01

High-quality Ge nanostructures are obtained by molecular beam epitaxy of Ge on Si(001) substrates at 200 °C and ex situ annealing at 400 °C. Their structural properties are comprehensively characterized by atomic force microscopy, transmission electron microscopy and Raman spectroscopy. It is disclosed that they are almost defect free except for some defects at the Ge/Si interface and in the subsequent Si capping layer. The misfit strain in the nanostructure is substantially relaxed. Dramatically strong photoluminescence (PL) from the Ge nanostructures is observed. Detailed analyses on the power- and temperature-dependent PL spectra, together with a self-consistent calculation, indicate the confinement and the high quantum efficiency of excitons within the Ge nanostructures. Our results demonstrate that the Ge nanostructures obtained via the present feasible route may have great potential in optoelectronic devices for monolithic optical-electronic integration circuits.

Synthesis and characterization of nanocrystalline Co-Fe-Nb-Ta-B alloy

NASA Astrophysics Data System (ADS)

Raanaei, Hossein; Fakhraee, Morteza

2017-09-01

In this research work, structural and magnetic evolution of Co57Fe13Nb8Ta4B18 alloy, during mechanical alloying process, have been investigated by using, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, electron dispersive X-ray spectroscopy, differential thermal analysis and also vibrating sample magnetometer. It is observed that at 120 milling time, the crystallite size reaches to about 7.8 nm. Structural analyses show that, the solid solution of the initial powder mixture occurs at160 h milling time. The coercivity behavior demonstrates a rise, up to 70 h followed by decreasing tendency up to final stage of milling process. Thermal analysis of 160 h milling time sample reveals two endothermic peaks. The characterization of annealed milled sample for 160 h milling time at 427 °C shows crystallite size growth accompanied by increasing in saturation magnetization.

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

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

Herpes simplex virus 1 induces egress channels through marginalized host chromatin

DOE PAGES

Myllys, Markko; Ruokolainen, Visa; Aho, Vesa; ...

2016-06-28

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. Here, we used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gapsmore » frequently contained viral nucleocapsids. Our results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.« less

Application of micro-computed tomography to microstructure studies of the medicinal fungus Hericium coralloides.

PubMed

Pallua, Johannes D; Kuhn, Volker; Pallua, Anton F; Pfaller, Kristian; Pallua, Anton K; Recheis, Wolfgang; Pöder, Reinhold

2015-01-01

The potential of 3-D nondestructive imaging techniques such as micro-computed tomography (micro-CT) was evaluated to study morphological patterns of the potential medicinal fungus Hericium coralloides (Basidiomycota). Micro-CT results were correlated with histological information gained from scanning electron microscopy (SEM) and light microscopy (LM). It is demonstrated that the combination of these imaging methods results in a more distinct picture of the morphology of the edible and potentially medicinal Hericium coralloides basidiomata. In addition we have created 3-D reconstructions and visualizations based on micro-CT imagery from a randomly selected part of the upper region of a fresh H. coralloides basidioma: Analyses for the first time allowed an approximation of the evolutionary effectiveness of this bizarrely formed basidioma type in terms of the investment of tissue biomass and its reproductive output (production of basidiospores). © 2015 by The Mycological Society of America.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.

PubMed

Aase, Audun; Hajdusek, Ondrej; Øines, Øivind; Quarsten, Hanne; Wilhelmsson, Peter; Herstad, Tove K; Kjelland, Vivian; Sima, Radek; Jalovecka, Marie; Lindgren, Per-Eric; Aaberge, Ingeborg S

2016-01-01

A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group. Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory. Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods. The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy

PubMed Central

Kim, Dahan; Curthoys, Nikki M.; Parent, Matthew T.; Hess, Samuel T.

2015-01-01

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined. PMID:26185614

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy.

PubMed

Kim, Dahan; Curthoys, Nikki M; Parent, Matthew T; Hess, Samuel T

2013-09-01

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined.

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.

Translational Immuno- and Neuro-imaging Demonstrate Corneal Neuro-immune Crosstalk

PubMed Central

Hamrah, Pedram; Seyed-Razavi, Yashar; Yamaguchi, Takefumi

2017-01-01

Corneal immuno- and neuro-imaging approaches facilitate in vivo analyses of the cornea, including high-resolution imaging of corneal immune cells and nerves. This approach facilitates the analyses of underlying immune and nerve alterations not detected by clinical slit-lamp examination alone. In this review, we describe recent work performed in our translational ocular immunology center with a focus on ‘bench-to-bedside’ and ‘bedside-to-bench’ research. The ability to visualize dendritiform immune cells (DCs) in patients with laser in vivo confocal microscopy (IVCM), recently discovered in the central murine cornea, has allowed us to demonstrated their utility as a potential surrogate biomarker for inflammatory ocular surface diseases. This biomarker for inflammation allows the measurement of therapeutic efficacy of anti-inflammatory drugs and its utility as an endpoint in clinical trials with high inter-observer agreement. IVCM image analyses from our studies demonstrated a significant increase in DC density and size in ocular disease, a positive correlation between DC density and clinical signs and symptoms of disease and pro-inflammatory tear cytokines, and a strong negative correlation between DC density and subbasal nerve density. In conjunction with pre-clinical research investigating the inflammatory state in a partial or fully denervated cornea, our results indicated that corneal nerves are directly involved in the regulation of homeostasis and immune privilege in the cornea. PMID:27631352

On the retrieval of crystallographic information from atom probe microscopy data via signal mapping from the detector coordinate space.

PubMed

Wallace, Nathan D; Ceguerra, Anna V; Breen, Andrew J; Ringer, Simon P

2018-06-01

Atom probe tomography is a powerful microscopy technique capable of reconstructing the 3D position and chemical identity of millions of atoms within engineering materials, at the atomic level. Crystallographic information contained within the data is particularly valuable for the purposes of reconstruction calibration and grain boundary analysis. Typically, analysing this data is a manual, time-consuming and error prone process. In many cases, the crystallographic signal is so weak that it is difficult to detect at all. In this study, a new automated signal processing methodology is demonstrated. We use the affine properties of the detector coordinate space, or the 'detector stack', as the basis for our calculations. The methodological framework and the visualisation tools are shown to be superior to the standard method of crystallographic pole visualisation directly from field evaporation images and there is no requirement for iterations between a full real-space initial tomographic reconstruction and the detector stack. The mapping approaches are demonstrated for aluminium, tungsten, magnesium and molybdenum. Implications for reconstruction calibration, accuracy of crystallographic measurements, reliability and repeatability are discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Growth of ferroelectric Ba{sub 0.8}Sr{sub 0.2}TiO{sub 3} epitaxial films by ultraviolet pulsed laser irradiation of chemical solution derived precursor layers

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

Queraltó, A.; Pérez del Pino, A., E-mail: aperez@icmab.es; Mata, M. de la

2015-06-29

Highly crystalline epitaxial Ba{sub 0.8}Sr{sub 0.2}TiO{sub 3} (BST) thin-films are grown on (001)-oriented LaNiO{sub 3}-buffered LaAlO{sub 3} substrates by pulsed laser irradiation of solution derived barium-zirconium-titanium precursor layers using a UV Nd:YAG laser source at atmospheric conditions. The structural analyses of the obtained films, studied by X-ray diffractometry and transmission electron microscopy, demonstrate that laser processing allows the growth of tens of nm-thick BST epitaxial films with crystalline structure similar to that of films obtained through conventional thermal annealing methods. However, the fast pulsed nature of the laser employed leads to crystallization kinetic evolution orders of magnitude faster than inmore » thermal treatments. The combination of specific photothermal and photochemical mechanisms is the main responsible for the ultrafast epitaxial laser-induced crystallization. Piezoresponse microscopy measurements demonstrate equivalent ferroelectric behavior in laser and thermally annealed films, being the piezoelectric constant ∼25 pm V{sup −1}.« less

Investigation of the Geochemical Preservation of ca. 3.0 Ga Permineralized and Encapsulated Microfossils by Nanoscale Secondary Ion Mass Spectrometry

NASA Astrophysics Data System (ADS)

Delarue, Frédéric; Robert, François; Sugitani, Kenichiro; Tartèse, Romain; Duhamel, Rémi; Derenne, Sylvie

2017-12-01

Observations of Archean organic-walled microfossils suggest that their fossilization took place through both encapsulation and permineralization. In this study, we investigated microfossils from the ca. 3.0 Ga Farrel Quartzite (Pilbara, Western Australia) using transmitted light microscopy, scanning electron microscopy, Raman microspectrometry, and nanoscale secondary ion mass spectrometry (NanoSIMS) ion microprobe analyses. In contrast to previous studies, we demonstrated that permineralized microfossils were not characterized by the micrometric spatial relationships between Si and C-N as observed in thin sections. Permineralized microfossils are composed of carbonaceous globules that did not survive the acid treatment, whereas encapsulated microfossils were characterized due to their resistance to the acid maceration procedure. We also investigated the microscale relationship between the 12C14N- and 12C2- ion emission as a proxy of the N/C atomic ratio in both permineralized and encapsulated microfossils. After considering any potential matrix and microtopography effects, we demonstrate that the encapsulated microfossils exhibit the highest level of geochemical preservation. This finding shows that the chemical heterogeneity of the microfossils, observed at a spatial resolution of a few hundreds of micrometers, can be related to fossilization processes.

Reactive Melt Extrusion To Improve the Dissolution Performance and Physical Stability of Naproxen Amorphous Solid Dispersions.

PubMed

Liu, Xu; Zhou, Lin; Zhang, Feng

2017-03-06

The purpose of this study was to investigate the reaction between naproxen (NPX) and meglumine (MEG) at elevated temperature and to study the effect of this reaction on the physical stabilities and in vitro drug-release properties of melt-extruded naproxen amorphous solid dispersions (ASDs). Differential scanning calorimetry, hot-stage polarized light microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy analyses demonstrated that in situ salt formation with proton transfer between NPX and MEG occurred at elevated temperature during the melt extrusion process. The amorphous NPX-MEG salt was physically most stable when two components were present at a 1:1 molar ratio. Polymeric carriers, including povidone, copovidone, and SOLUPLUS, did not interfere with the reaction between NPX and MEG during melt extrusion. Compared to the traditional NPX ASDs consisting of NPX and polymer only, NPX-MEG ASDs were physically more stable and remained amorphous following four months storage at 40 °C and 75% RH (relative humidity). Based on nonsink dissolution testing and polarized light microscopy analyses, we concluded that the conventional NPX ASDs composed of NPX and polymers failed to improve the NPX dissolution rate due to the rapid recrystallization of NPX in contact with aqueous medium. The dissolution rate of NPX-MEG ASDs was two times greater than the corresponding physical mixtures and conventional NPX ASDs. This study demonstrated that the acid-base reaction between NPX and MEG during melt extrusion significantly improved the physical stability and the dissolution rate of NPX ASDs.

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.

Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles.

PubMed

Martin, Jessica L; Cao, Sheng; Maldonado, Jose O; Zhang, Wei; Mansky, Louis M

2016-09-15

The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta- and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta- and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

PubMed

Chakraborty, Nilay; Wang, Mian; Solocinski, Jason; Kim, Wonsuk; Argento, Alan

2016-01-01

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling

NASA Astrophysics Data System (ADS)

Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D.; Sweedler, Jonathan V.

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. [Figure not available: see fulltext.

microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling.

PubMed

Comi, Troy J; Neumann, Elizabeth K; Do, Thanh D; Sweedler, Jonathan V

2017-09-01

Image-guided mass spectrometry (MS) profiling provides a facile framework for analyzing samples ranging from single cells to tissue sections. The fundamental workflow utilizes a whole-slide microscopy image to select targets of interest, determine their spatial locations, and subsequently perform MS analysis at those locations. Improving upon prior reported methodology, a software package was developed for working with microscopy images. microMS, for microscopy-guided mass spectrometry, allows the user to select and profile diverse samples using a variety of target patterns and mass analyzers. Written in Python, the program provides an intuitive graphical user interface to simplify image-guided MS for novice users. The class hierarchy of instrument interactions permits integration of new MS systems while retaining the feature-rich image analysis framework. microMS is a versatile platform for performing targeted profiling experiments using a series of mass spectrometers. The flexibility in mass analyzers greatly simplifies serial analyses of the same targets by different instruments. The current capabilities of microMS are presented, and its application for off-line analysis of single cells on three distinct instruments is demonstrated. The software has been made freely available for research purposes. Graphical Abstract ᅟ.

HAADF-STEM atom counting in atom probe tomography specimens: Towards quantitative correlative microscopy.

PubMed

Lefebvre, W; Hernandez-Maldonado, D; Moyon, F; Cuvilly, F; Vaudolon, C; Shinde, D; Vurpillot, F

2015-12-01

The geometry of atom probe tomography tips strongly differs from standard scanning transmission electron microscopy foils. Whereas the later are rather flat and thin (<20 nm), tips display a curved surface and a significantly larger thickness. As far as a correlative approach aims at analysing the same specimen by both techniques, it is mandatory to explore the limits and advantages imposed by the particular geometry of atom probe tomography specimens. Based on simulations (electron probe propagation and image simulations), the possibility to apply quantitative high angle annular dark field scanning transmission electron microscopy to of atom probe tomography specimens has been tested. The influence of electron probe convergence and the benefice of deconvolution of electron probe point spread function electron have been established. Atom counting in atom probe tomography specimens is for the first time reported in this present work. It is demonstrated that, based on single projections of high angle annular dark field imaging, significant quantitative information can be used as additional input for refining the data obtained by correlative analysis of the specimen in APT, therefore opening new perspectives in the field of atomic scale tomography. Copyright © 2015 Elsevier B.V. All rights reserved.

Diffusion and Binding of Laponite Clay Nanoparticles into Collagen Fibers for the Formation of Leather Matrix.

PubMed

Shi, Jiabo; Wang, Chunhua; Ngai, To; Lin, Wei

2018-06-13

Understanding accessibility and interactions of clay nanoparticles with collagen fibers is an important fundamental issue for the conversion of collagen to leather matrix. In this study, we have investigated the diffusion and binding of Laponite into the collagen fiber network. Our results indicate that the diffusion behaviors of Laponite into the collagen exhibit the Langmuir adsorption, verifying its affinity for collagen. The introduction of Laponite leads to a shift in the isoelectric point of collagen from ∼6.8 to ∼4.5, indicating the ionic bonding between the positively charged amino groups of the collagen and negatively charged Laponite under the tanning conditions. Fluorescence microscopy, atomic force microscopy, field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and wide-angle X-ray diffraction analyses reveal that Laponite nanoparticles can penetrate into collagen microstructure and evenly distributed onto collagen fibrils, not altering native D-periodic banding patterns of collagen fibrils. Attenuated total reflectance-Fourier transform infrared and Raman spectroscopy detections further demonstrate the presence of noncovalent interactions, namely, ionic and hydrogen bonding, between Laponite and collagen. These findings provide a theoretical basis for the use of Laponite as an emerging tanning agent in leather manufacture.

Theoretical study of carbon-based tips for scanning tunnelling microscopy.

PubMed

González, C; Abad, E; Dappe, Y J; Cuevas, J C

2016-03-11

Motivated by recent experiments, we present here a detailed theoretical analysis of the use of carbon-based conductive tips in scanning tunnelling microscopy. In particular, we employ ab initio methods based on density functional theory to explore a graphitic, an amorphous carbon and two diamond-like tips for imaging with a scanning tunnelling microscope (STM), and we compare them with standard metallic tips made of gold and tungsten. We investigate the performance of these tips in terms of the corrugation of the STM images acquired when scanning a single graphene sheet. Moreover, we analyse the impact of the tip-sample distance and show that it plays a fundamental role in the resolution and symmetry of the STM images. We also explore in depth how the adsorption of single atoms and molecules in the tip apexes modifies the STM images and demonstrate that, in general, it leads to an improved image resolution. The ensemble of our results provides strong evidence that carbon-based tips can significantly improve the resolution of STM images, as compared to more standard metallic tips, which may open a new line of research in scanning tunnelling microscopy.

Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

PubMed

Cook, Michael A; Chan, Chi-Kin; Jorgensen, Paul; Ketela, Troy; So, Daniel; Tyers, Mike; Ho, Chi-Yip

2008-02-06

Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base) unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

An integrated approach to the Taxonomic identification of prehistoric shell ornaments

USGS Publications Warehouse

Demarchi, Beatrice; O'Connor, Sonia; Ponzoni, Andre de Lima; Ponzoni, Raquel de Almeida Roch; Sheridan, Alison; Penkman, Kirsty; Hancock, Y.; Wilson, Julie

2014-01-01

Shell beads appear to have been one of the earliest examples of personal adornments. Marine shells identified far from the shore evidence long-distance transport and imply networks of exchange and negotiation. However, worked beads lose taxonomic clues to identification, and this may be compounded by taphonomic alteration. Consequently, the significance of this key early artefact may be underestimated. We report the use of bulk amino acid composition of the stable intra-crystalline proteins preserved in shell biominerals and the application of pattern recognition methods to a large dataset (777 samples) to demonstrate that taxonomic identification can be achieved at genus level. Amino acid analyses are fast (<2 hours per sample) and micro-destructive (sample size <2 mg). Their integration with non-destructive techniques provides a valuable and affordable tool, which can be used by archaeologists and museum curators to gain insight into early exploitation of natural resources by humans. Here we combine amino acid analyses, macro- and microstructural observations (by light microscopy and scanning electron microscopy) and Raman spectroscopy to try to identify the raw material used for beads discovered at the Early Bronze Age site of Great Cornard (UK). Our results show that at least two shell taxa were used and we hypothesise that these were sourced locally.

Uranium association with iron-bearing phases in mill tailings from Gunnar, Canada.

PubMed

Othmane, Guillaume; Allard, Thierry; Morin, Guillaume; Sélo, Madeleine; Brest, Jessica; Llorens, Isabelle; Chen, Ning; Bargar, John R; Fayek, Mostafa; Calas, Georges

2013-11-19

The speciation of uranium was studied in the mill tailings of the Gunnar uranium mine (Saskatchewan, Canada), which operated in the 1950s and 1960s. The nature, quantification, and spatial distribution of uranium-bearing phases were investigated by chemical and mineralogical analyses, fission track mapping, electron microscopy, and X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopies at the U LIII-edge and Fe K-edge. In addition to uranium-containing phases from the ore, uranium is mostly associated with iron-bearing minerals in all tailing sites. XANES and EXAFS data and transmission electron microscopy analyses of the samples with the highest uranium concentrations (∼400-700 mg kg(-1) of U) demonstrate that uranium primarily occurs as monomeric uranyl ions (UO2(2+)), forming inner-sphere surface complexes bound to ferrihydrite (50-70% of the total U) and to a lesser extent to chlorite (30-40% of the total U). Thus, the stability and mobility of uranium at the Gunnar site are mainly influenced by sorption/desorption processes. In this context, acidic pH or alkaline pH with the presence of UO2(2+)- and/or Fe(3+)-complexing agents (e.g., carbonate) could potentially solubilize U in the tailings pore waters.

Bat white-nose syndrome: An emerging fungal pathogen?

USGS Publications Warehouse

Blehert, D.S.; Hicks, A.C.; Behr, M.; Meteyer, C.U.; Berlowski-Zier, B. M.; Buckles, E.L.; Coleman, J.T.H.; Darling, S.R.; Gargas, A.; Niver, R.; Okoniewski, J.C.; Rudd, R.J.; Stone, W.B.

2009-01-01

White-nose syndrome (WNS) is a condition associated with an unprecedented bat mortality event in the northeastern United States. Since the winter of 2006*2007, bat declines exceeding 75% have been observed at surveyed hibernacula. Affected bats often present with visually striking white fungal growth on their muzzles, ears, and/or wing membranes. Direct microscopy and culture analyses demonstrated that the skin of WNS-affected bats is colonized by a psychro-philic fungus that is phylogenetically related to Geomyces spp. but with a conidial morphology distinct from characterized members of this genus. This report characterizes the cutaneous fungal infection associated with WNS.

High-resolution structural and elemental analyses of calcium storage structures synthesized by the noble crayfish Astacus astacus.

PubMed

Luquet, Gilles; Salomé, Murielle; Ziegler, Andreas; Paris, Céline; Percot, Aline; Dauphin, Yannicke

2016-11-01

During premolt, crayfish develop deposits of calcium ions, called gastroliths, in their stomach wall. The stored calcium is used for the calcification of parts of the skeleton regularly renewed for allowing growth. Structural and molecular analyses of gastroliths have been primarily performed on three crayfish species, Orconectes virilis, Procambarus clarkii, and more recently, Cherax quadricarinatus. We have performed high-resolution analyses of gastroliths from the native noble crayfish, Astacus astacus, focusing on the microstructure, the mineralogical and elemental composition and distribution in a comparative perspective. Field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) observations showed a classical layered microstructure composed of 200-nm diameter granules aligned along fibers. These granules are themselves composed of agglomerated nanogranules of 50nm-mean diameters. Denser regions of bigger fused granules are also present. Micro-Raman spectroscopy show that if A. astacus gastroliths, similarly to the other analyzed gastroliths, are mainly composed of amorphous calcium carbonate (ACC), they are also rich in amorphous calcium phosphate (ACP). The presence of a carotenoid pigment is also observed in A. astacus gastrolith contrary to C. quadricarinatus. Energy-dispersive X-ray spectroscopy (EDX) analyses demonstrate the presence of minor elements such as Mg, Sr, Si and P. The distribution of this last element is particularly heterogeneous. X-ray absorption near edge structure spectroscopy (XANES) reveals an alternation of layers more or less rich in phosphorus evidenced in the mineral phase as well as in the organic matrix in different molecular forms. Putative functions of the different P-comprising molecules are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential protein folding and chemical changes in lung tissues exposed to asbestos or particulates

PubMed Central

Pascolo, Lorella; Borelli, Violetta; Canzonieri, Vincenzo; Gianoncelli, Alessandra; Birarda, Giovanni; Bedolla, Diana E.; Salomé, Murielle; Vaccari, Lisa; Calligaro, Carla; Cotte, Marine; Hesse, Bernhard; Luisi, Fernando; Zabucchi, Giuliano; Melato, Mauro; Rizzardi, Clara

2015-01-01

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (μXRF) and Fourier Transform InfraRed (μFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. μXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. μFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of β-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects. PMID:26159651

Evaluation of morpho-anatomical and chemical differences between varieties of the medicinal plant Casearia sylvestris Swartz.

PubMed

Claudino, Josiane C; Sacramento, Luis V S do; Koch, Ingrid; Santos, Helen A; Cavalheiro, Alberto J; Tininis, Aristeu G; Santos, André G dos

2013-01-01

Casearia sylvestris Swartz (Salicaceae) has been used in traditional medicine and its leaf extracts have been exhibited important pharmacological activities. The species presents morphological, chemical and genetic variation. Two varieties are considered due external morphological differences: C. sylvestris var. sylvestris and var. lingua. There are difficulties in definition of these varieties. The objective of this work is to evaluate chemical and morpho-anatomical differences between C. sylvestris varieties that can be applied in their distinction for pharmaceutical or botanical purposes. Transverse and paradermic sections of leaves were prepared for morpho-anatomical, histochemical and quantitative microscopy (stomatal and palisade index) analyses. Diterpene profiles of the specimens were obtained by HPLC-DAD and TLC. Morpho-anatomical analyses demonstrated significant differences between the varieties only in paradermic sections: var. sylvestris--polygonal epidermic cell walls and hypostomatic; var. lingua--rounded epidermic cell walls and amphistomatic. No differences were observed for stomatal index; palisade index was found 2.8 for var. lingua and 3.9 for var. sylvestris. Chromatographic analyses confirmed previous results demonstrating that diterpene profile in varieties differs, with predominance of these metabolites in var. sylvestris. In conclusion, this work indicates that chromatographic analysis besides morpho-anatomical analysis can be applied in distinction of C. sylvestris varieties.

Publications - GMC 58 | Alaska Division of Geological & Geophysical Surveys

Science.gov Websites

DGGS GMC 58 Publication Details Title: X-ray diffraction and scanning electron microscopy mineral , Michael, and Core Laboratories, 1985, X-ray diffraction and scanning electron microscopy mineral analyses

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

Size-selective breaking of the core-shell structure of gallium nanoparticles.

PubMed

Catalán Gómez, Sergio; Redondo-Cubero, Andres; Palomares Simon, Francisco Javier; Vazquez Burgos, Luis; Nogales, Emilio; Nucciarelli, Flavio; Mendez, Bianchi; Gordillo, Nuria; Pau, Jose Luis

2018-06-11

Core-shell gallium nanoparticles (Ga NPs) have recently been proposed as an ultraviolet plasmonic material for different applications but only at room temperature. Here, the thermal stability as a function of the size of the NPs is reported over a wide range of temperatures. We analyse the chemical and structural properties of the oxide shell by x-ray photoelectron spectroscopy and atomic force microscopy. We demonstrate the inverse dependence of the shell breaking temperature with the size of the NPs. Spectroscopic ellipsometry is used for tracking the rupture and its mechanism is systematically investigated by scanning electron microscopy, grazing incidence x-ray diffraction and cathodoluminescence. Taking advantage of the thermal stability of the NPs, we perform complete oxidations that lead to homogenous gallium oxide NPs. Thus, this study set the physical limits of Ga NPs to last at high temperatures, and opens up the possibility to achieve totally oxidized NPs while keeping their sphericity. © 2018 IOP Publishing Ltd.

Scanning transmission ion micro-tomography (STIM-T) of biological specimens.

PubMed

Schwertner, Micheal; Sakellariou, Arthur; Reinert, Tilo; Butz, Tilman

2006-05-01

Computed tomography (CT) was applied to sets of Scanning Transmission Ion Microscopy (STIM) projections recorded at the LIPSION ion beam laboratory (Leipzig) in order to visualize the 3D-mass distribution in several specimens. Examples for a test structure (copper grid) and for biological specimens (cartilage cells, cygospore) are shown. Scanning Transmission Micro-Tomography (STIM-T) at a resolution of 260 nm was demonstrated for the first time. Sub-micron features of the Cu-grid specimen were verified by scanning electron microscopy. The ion energy loss measured during a STIM-T experiment is related to the mass density of the specimen. Typically, biological specimens can be analysed without staining. Only shock freezing and freeze-drying is required to preserve the ultra-structure of the specimen. The radiation damage to the specimen during the experiment can be neglected. This is an advantage compared to other techniques like X-ray micro-tomography. At present, the spatial resolution is limited by beam position fluctuations and specimen vibrations.

Green chemistry approach for the synthesis of ZnO-carbon dots nanocomposites with good photocatalytic properties under visible light.

PubMed

Bozetine, Hakima; Wang, Qi; Barras, Alexandre; Li, Musen; Hadjersi, Toufik; Szunerits, Sabine; Boukherroub, Rabah

2016-03-01

We report on a simple and one-pot synthetic method to produce ZnO/carbon quantum dots (ZnO/CQDs) nanocomposites. The morphological features and chemical composition of the nanocomposites were characterized using X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analyses (TGA), X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The optical properties of the nanocomposites were examined using UV-visible (UV-vis) spectrophotometry. The photocatalytic activity of the ZnO/CQDs was evaluated for the degradation of a model organic pollutant, rhodamine B, under visible light irradiation at room temperature. The highly efficient photodegradation capability of the nanocomposite was demonstrated by comparison with ZnO particles, prepared using identical experimental conditions. Overall, the present approach adheres to green chemistry principles and the nanocomposite holds promise for the development of remarkably efficient catalytic systems. Copyright © 2015 Elsevier Inc. All rights reserved.

Biosynthesis of silver nanoparticles using aqueous leaf extract of Thevetia peruviana Juss and its antimicrobial activities

NASA Astrophysics Data System (ADS)

Oluwaniyi, Omolara O.; Adegoke, Haleemat I.; Adesuji, Elijah T.; Alabi, Aderemi B.; Bodede, Sunday O.; Labulo, Ayomide H.; Oseghale, Charles O.

2016-08-01

Biosynthesizing of silver nanoparticles using microorganisms or various plant parts have proven more environmental friendly, cost-effective, energy saving and reproducible when compared to chemical and physical methods. This investigation demonstrated the plant-mediated synthesis of silver nanoparticles using the aqueous leaf extract of Thevetia peruviana. UV-Visible spectrophotometer was used to measure the surface plasmon resonance of the nanoparticles at 460 nm. Fourier Transform Infrared showed that the glycosidic -OH and carbonyl functional group present in extract were responsible for the reduction and stabilization of the silver nanoparticles. X ray diffraction, Scanning Electron Microscopy, Transmission Electron Microscopy and Selected Area Electron Diffraction analyses were used to confirm the nature, morphology and shape of the nanoparticles. The silver nanoparticles are spherical in shape with average size of 18.1 nm. The synthesized silver nanoparticles showed activity against fungal pathogens and bacteria. The zone of inhibition observed in the antimicrobial study ranged between 10 and 20 mm.

Different molecular organization of two carotenoids, lutein and zeaxanthin, in human colon epithelial cells and colon adenocarcinoma cells

NASA Astrophysics Data System (ADS)

Grudzinski, Wojciech; Piet, Mateusz; Luchowski, Rafal; Reszczynska, Emilia; Welc, Renata; Paduch, Roman; Gruszecki, Wieslaw I.

2018-01-01

Two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of exogenous carotenoids, either zeaxanthin or lutein. Both carotenoids demonstrated cytotoxicity with respect to cancer cells but not to normal cells. Cells from both the cell lines were analyzed with application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. Both imaging techniques show effective incorporation of carotenoid molecules into growing cells. Comparison of the Raman scattering and fluorescence lifetime characteristics reveals different molecular organization of carotenoids in the carcinoma and normal cells. The main difference consists in a carotenoid aggregation level which is substantially lower in the carcinoma cells as compared to the normal cells. Different molecular organization of carotenoids was interpreted in terms of a different metabolism of normal and carcinoma cells and has been concluded to provide a possibility of cancer diagnosis based on spectroscopic analyses.

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis

PubMed Central

Kozai, Toshiya; Yang, Huiran; Ishikuro, Daiki; Seyama, Kaho; Kumagai, Yusuke; Abe, Tadashi; Yamada, Hiroshi; Uchihashi, Takayuki

2018-01-01

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission. PMID:29357276

Myoanatomy of the velvet worm leg revealed by laboratory-based nanofocus X-ray source tomography.

PubMed

Müller, Mark; de Sena Oliveira, Ivo; Allner, Sebastian; Ferstl, Simone; Bidola, Pidassa; Mechlem, Korbinian; Fehringer, Andreas; Hehn, Lorenz; Dierolf, Martin; Achterhold, Klaus; Gleich, Bernhard; Hammel, Jörg U; Jahn, Henry; Mayer, Georg; Pfeiffer, Franz

2017-11-21

X-ray computed tomography (CT) is a powerful noninvasive technique for investigating the inner structure of objects and organisms. However, the resolution of laboratory CT systems is typically limited to the micrometer range. In this paper, we present a table-top nanoCT system in conjunction with standard processing tools that is able to routinely reach resolutions down to 100 nm without using X-ray optics. We demonstrate its potential for biological investigations by imaging a walking appendage of Euperipatoides rowelli , a representative of Onychophora-an invertebrate group pivotal for understanding animal evolution. Comparative analyses proved that the nanoCT can depict the external morphology of the limb with an image quality similar to scanning electron microscopy, while simultaneously visualizing internal muscular structures at higher resolutions than confocal laser scanning microscopy. The obtained nanoCT data revealed hitherto unknown aspects of the onychophoran limb musculature, enabling the 3D reconstruction of individual muscle fibers, which was previously impossible using any laboratory-based imaging technique.

Dimension- and shape-dependent thermal transport in nano-patterned thin films investigated by scanning thermal microscopy

NASA Astrophysics Data System (ADS)

Ge, Yunfei; Zhang, Yuan; Weaver, Jonathan M. R.; Dobson, Phillip S.

2017-12-01

Scanning thermal microscopy (SThM) is a technique which is often used for the measurement of the thermal conductivity of materials at the nanometre scale. The impact of nano-scale feature size and shape on apparent thermal conductivity, as measured using SThM, has been investigated. To achieve this, our recently developed topography-free samples with 200 and 400 nm wide gold wires (50 nm thick) of length of 400-2500 nm were fabricated and their thermal resistance measured and analysed. This data was used in the development and validation of a rigorous but simple heat transfer model that describes a nanoscopic contact to an object with finite shape and size. This model, in combination with a recently proposed thermal resistance network, was then used to calculate the SThM probe signal obtained by measuring these features. These calculated values closely matched the experimental results obtained from the topography-free sample. By using the model to analyse the dimensional dependence of thermal resistance, we demonstrate that feature size and shape has a significant impact on measured thermal properties that can result in a misinterpretation of material thermal conductivity. In the case of a gold nanowire embedded within a silicon nitride matrix it is found that the apparent thermal conductivity of the wire appears to be depressed by a factor of twenty from the true value. These results clearly demonstrate the importance of knowing both probe-sample thermal interactions and feature dimensions as well as shape when using SThM to quantify material thermal properties. Finally, the new model is used to identify the heat flux sensitivity, as well as the effective contact size of the conventional SThM system used in this study.

A novel multiplex bead-based platform highlights the diversity of extracellular vesicles

PubMed Central

Koliha, Nina; Wiencek, Yvonne; Heider, Ute; Jüngst, Christian; Kladt, Nikolay; Krauthäuser, Susanne; Johnston, Ian C. D.; Bosio, Andreas; Schauss, Astrid; Wild, Stefan

2016-01-01

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions. PMID:26901056

In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

NASA Astrophysics Data System (ADS)

Stuhlmüller, M.; Schwarz-Finsterle, J.; Fey, E.; Lux, J.; Bach, M.; Cremer, C.; Hinderhofer, K.; Hausmann, M.; Hildenbrand, G.

2015-10-01

Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

Mode of action studies on the formation of enamel minerals from a novel toothpaste containing calcium silicate and sodium phosphate salts.

PubMed

Sun, Yuekui; Li, Xiaoke; Deng, Yan; Sun, Jianing N; Tao, DanYing; Chen, Hui; Hu, Qinghong; Liu, Renjiang; Liu, Weining; Feng, Xiping; Wang, Jinfang; Carvell, Mel; Joiner, Andrew

2014-06-01

To investigate in vitro and in situ the deposition and formation of hydroxyapatite (HAP) on enamel surfaces following brushing with a novel toothpaste containing calcium silicate (CaSi), sodium phosphate salts and fluoride. Polished enamel blocks were brushed in vitro with a slurry of the CaSi toothpaste. After one brush and four weeks simulated brushing the enamel surfaces were analysed. In an in situ protocol, enamel blocks were attached to first or second molar teeth of healthy subjects, exposed to 4 weeks twice per day brushing with the CaSi toothpaste and then analysed. The surface deposits were analysed using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). In addition, the CaSi toothpaste was slurried in simulated oral fluid (SOF) over a 3 hour period and the solids were isolated and analysed by Fourier transform infrared spectroscopy (FTIR). The FTIR study demonstrated that calcium phosphate phases had formed and these became increasingly crystalline over 3 hours. CaSi was deposited onto enamel surfaces following one brushing with the toothpaste in vitro.The deposited particles showed evidence of HAP crystalline phases associated with the CaSi. Following 4 weeks brushing in vitro, the deposition increased and analyses showed that the deposited material was HAP. These results were confirmed by the in situ study. Calcium silicate can be deposited onto enamel surfaces from a novel toothpaste formulation where it can form the enamel mineral HAP. A novel toothpaste formulation containing CaSi can form HAP on enamel surfaces. The potential of this technology is for a novel approach to the repair of demineralised enamel and the protection of enamel during acid exposure. © 2014 Elsevier Ltd. All rights reserved.

Flower-like NiO structures: Controlled hydrothermal synthesis and electrochemical characteristic

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

Chai, Hui; Chen, Xuan; Key Laboratory of Advanced Functional Materials, Institute of Applied Chemistry, Xinjiang University, Urumqi 830046, Xinjiang

Graphical abstract: Flower-like porous NiO was obtained via thermal decomposition of the precursor prepared by a hydrothermal process using hexamethylenetetramine and polyethylene glycol as hydrolysis-controlling agent and surfactant, respectively. The morphology and microstructure of as-synthesized NiO were characterized by X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of electrochemical measurements demonstrated that the flower-like porous NiO has high capacity (340 F g{sup −1}) with excellent cycling performance as electrode materials of electrochemical capacitors (ECs), which may be attributed to the unique microstrcture of NiO. Data analyses indicated that NiO with novel porousmore » structure attractive for practical and large-scale applications in electrochemical capacitors. Display Omitted Highlights: ► Synthesis and characterization of NiO with novel porous structure is presented in this work. ► The electrochemical performance of product was examined. ► NiO with excellent performance as electrode materials may be due to the unique microstrcture. ► NiO with novel porous structure attractive for practical with high capacity (340 F g{sup −1}). -- Abstract: Flower-like porous NiO was obtained by thermal decomposition of the precursor prepared by a hydrothermal process with hexamethylenetetramine and polyethylene glycol as hydrolysis-controlling agent and surfactant, respectively. The morphology and microstructure of as-synthesized NiO were characterized by X-ray diffraction (XRD), Brunauer–Emmett–Teller (BET), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The resulting structures of NiO exhibited porous like petal building blocks. The electrochemical measurements’ results demonstrated that flower-like porous NiO has high capacity (340 F g{sup −1}) with excellent cycling performance as electrode materials for electrochemical capacitors, which may be attributed to the unique structure of NiO. The results indicated that NiO with novel porous structure has been attractive for practical and large-scale applications in electrochemical capacitors.« less

Novel synthesis of ZnO/PMMA nanocomposites for photocatalytic applications

PubMed Central

Di Mauro, Alessandro; Cantarella, Maria; Nicotra, Giuseppe; Pellegrino, Giovanna; Gulino, Antonino; Brundo, Maria Violetta; Privitera, Vittorio; Impellizzeri, Giuliana

2017-01-01

The incorporation of nanostructured photocatalysts in polymers is a strategic way to obtain novel water purification systems. This approach takes the advantages of: (1) the presence of nanostructured photocatalyst; (2) the flexibility of polymer; (3) the immobilization of photocatalyst, that avoids the recovery of the nanoparticles after the water treatment. Here we present ZnO-polymer nanocomposites with high photocatalytic performance and stability. Poly (methyl methacrylate) (PMMA) powders were coated with a thin layer of ZnO (80 nm thick) by atomic layer deposition at low temperature (80 °C). Then the method of sonication and solution casting was performed so to obtain the ZnO/PMMA nanocomposites. A complete morphological, structural, and chemical characterization was made by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) analyses. The remarkable photocatalytic efficiency of the nanocomposites was demonstrated by the degradation of methylene blue (MB) dye and phenol in aqueous solution under UV light irradiation. The composites also resulted reusable and stable, since they maintained an unmodified photo-activity after several MB discoloration runs. Thus, these results demonstrate that the proposed ZnO/PMMA nanocomposite is a promising candidate for photocatalytic applications and, in particular, for novel water treatment. PMID:28098229

Magnetic resonance microscopy-based analyses of the neuroanatomical effects of gestational day 9 ethanol exposure in mice

PubMed Central

Parnell, Scott E.; Holloway, Hunter T.; O’Leary-Moore, Shonagh K.; Dehart, Deborah B.; Paniaqua, Beatriz; Oguz, Ipek; Budin, Francois; Styner, Martin A.; Johnson, G. Allan; Sulik, Kathleen K.

2013-01-01

Animal model-based studies have shown that ethanol exposure during early gestation induces developmental stage-specific abnormalities of the face and brain. The exposure time-dependent variability in ethanol’s teratogenic outcomes is expected to contribute significantly to the wide spectrum of effects observed in humans with fetal alcohol spectrum disorder (FASD). The work presented here employs a mouse FASD model and magnetic resonance microscopy (MRM; high resolution magnetic resonance imaging) in studies designed to further our understanding of the developmental stage-specific defects of the brain that are induced by ethanol. At neurulation stages, i.e. at the beginning of gestational day (GD) 9 and again 4 hours later, time-mated C57Bl/6J dams were intraperitoneally administered 2.9 g/kg ethanol or vehicle. Ethanol-exposed fetuses were collected on GD 17, processed for MRM analysis, and results compared to comparably staged controls. Linear and volume measurements as well as shape changes for numerous individual brain regions were determined. GD 9 ethanol exposure resulted in significantly increased septal region width, reduction of cerebellar volume, and enlargement of all of the ventricles. Additionally, the results of shape analyses showed that many areas of the ethanol-exposed brains including the cerebral cortex, hippocampus and right striatum were significantly misshapen. These data demonstrate that ethanol can induce dysmorphology that may not be obvious based on volumetric analyses alone, highlight the asymmetric aspects of ethanol-induced defects, and add to our understanding of ethanol’s developmental stage-dependent neuroteratogenesis. PMID:23911654

Traditional microscopy instruction versus process-oriented virtual microscopy instruction: a naturalistic experiment with control group.

PubMed

Helle, Laura; Nivala, Markus; Kronqvist, Pauliina; Gegenfurtner, Andreas; Björk, Pasi; Säljö, Roger

2011-03-30

Virtual microscopy is being introduced in medical education as an approach for learning how to interpret information in microscopic specimens. It is, however, far from evident how to incorporate its use into existing teaching practice. The aim of the study was to explore the consequences of introducing virtual microscopy tasks into an undergraduate pathology course in an attempt to render the instruction more process-oriented. The research questions were: 1) How is virtual microscopy perceived by students? 2) Does work on virtual microscopy tasks contribute to improvement in performance in microscopic pathology in comparison with attending assistant-led demonstrations only? During a one-week period, an experimental group completed three sets of virtual microscopy homework assignments in addition to attending demonstrations. A control group attended the demonstrations only. Performance in microscopic pathology was measured by a pre-test and a post-test. Student perceptions of regular instruction and virtual microscopy were collected one month later by administering the Inventory of Intrinsic Motivation and open-ended questions. The students voiced an appreciation for virtual microscopy for the purposes of the course and for self-study. As for learning gains, the results indicated that learning was speeded up in a subgroup of students consisting of conscientious high achievers. The enriched instruction model may be suited as such for elective courses following the basic course. However, the instructional model needs further development to be suited for basic courses.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

Systematic Validation and Atomic Force Microscopy of Non-Covalent Short Oligonucleotide Barcode Microarrays

PubMed Central

Cook, Michael A.; Chan, Chi-Kin; Jorgensen, Paul; Ketela, Troy; So, Daniel; Tyers, Mike; Ho, Chi-Yip

2008-01-01

Background Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20–60 base) unique sequence tags, or “barcodes”, associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. Methodology/Principal Findings Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5′-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM), we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. Conclusions/Significance These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis. PMID:18253494

Characterisation of the surface topography, tomography and chemistry of fretting corrosion product found on retrieved polished femoral stems.

PubMed

Bryant, M; Ward, M; Farrar, R; Freeman, R; Brummitt, K; Nolan, J; Neville, A

2014-04-01

This study presents the characterisation of the surface topography, tomography and chemistry of fretting corrosion product found on retrieved polished femoral stems. Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), Transmission Electron Microscopy (TEM) and Fourier Transform Infrared Spectroscopy (FI-IR) were utilised in order to assess the surface morphology of retrieved Metal-on-Metal Total Hip Replacements and surface chemistry of the films found on the surface. Gross slip, plastic deformation and directionality of the surface were extensively seen on the proximal surfaces of the retrievals. A more corrosive phenomenon was observed in the distal regions of the stem, demonstrating a seemingly intergranular attack. Tribochemical reactions were seen to occur within the stem-cement interfaces with tribofilms being observed on the femoral stem and counterpart PMMA bone cement. XPS, TEM-EDX and FT-IR analyses demonstrated that the films present in the stem surfaces were a complex mixture of chromium oxide and amorphous organic material. A comparison between current experimental and clinical literature has been conducted and findings from this study demonstrate that the formation and chemistry of films are drastically influenced by the type of wear or degradation mechanism. Films formed in the stem-cement interface are thought to further influence the biological environment outside the stem-cement interface due to the formation of Cr and O rich films within the interface whilst Co is free to migrate away. © 2013 Elsevier Ltd. All rights reserved.

Pitting corrosion of titanium

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

Casillas, N.; Charlebois, S.; Smyrl, W.H.

1994-03-01

The breakdown of native and anodically grown oxide films on Ti electrodes is investigated by scanning electrochemical microscopy (SECM), video microscopy, transmission electron microscopy, and voltammetry. SECM is used to demonstrated that the oxidation of Br[sup [minus

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders.

PubMed

Malfatti, Edoardo; Olivé, Montse; Taratuto, Ana Lía; Richard, Pascale; Brochier, Guy; Bitoun, Marc; Gueneau, Lucie; Laforêt, Pascal; Stojkovic, Tanya; Maisonobe, Thierry; Monges, Soledad; Lubieniecki, Fabiana; Vasquez, Gabriel; Streichenberger, Nathalie; Lacène, Emmanuelle; Saccoliti, Maria; Prudhon, Bernard; Alexianu, Marilena; Figarella-Branger, Dominique; Schessl, Joachim; Bonnemann, Carsten; Eymard, Bruno; Fardeau, Michel; Bonne, Gisèle; Romero, Norma Beatriz

2013-09-01

FHL1 mutations have been associated with various disorders that include reducing body myopathy (RBM), Emery-Dreifuss-like muscular dystrophy, isolated hypertrophic cardiomyopathy, and some overlapping conditions. We report a detailed histochemical, immunohistochemical, electron microscopic, and immunoelectron microscopic analyses of muscle biopsies from 18 patients carrying mutations in FHL1: 14 RBM patients (Group 1), 3 Emery-Dreifuss muscular dystrophy patients (Group 2), and 1 patient with hypertrophic cardiomyopathy and muscular hypertrophy (Group 2). Group 1 muscle biopsies consistently showed RBs associated with cytoplasmic bodies. The RBs showed prominent FHL1 immunoreactivity whereas desmin, αB-crystallin, and myotilin immunoreactivity surrounded RBs. By electron microscopy, RBs were composed of electron-dense tubulofilamentous material that seemed to spread progressively between the myofibrils and around myonuclei. By immunoelectron microscopy, FHL1 protein was found exclusively inside RBs. Group 2 biopsies showed mild dystrophic abnormalities without RBs; only minor nonspecific myofibrillar abnormalities were observed under electron microscopy. Molecular analysis revealed missense mutations in the second FHL1 LIM domain in Group 1 patients and ins/del or missense mutations within the fourth FHL1 LIM domain in Group 2 patients. Our findings expand the morphologic features of RBM, clearly demonstrate the localization of FHL1 in RBs, and further illustrate major morphologic differences among different FHL1-related myopathies.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

PubMed

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa E; Cuervo, Patricia; Jesus, José Batista de

2017-10-01

Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

A probabilistic approach to joint cell tracking and segmentation in high-throughput microscopy videos.

PubMed

Arbelle, Assaf; Reyes, Jose; Chen, Jia-Yun; Lahav, Galit; Riklin Raviv, Tammy

2018-04-22

We present a novel computational framework for the analysis of high-throughput microscopy videos of living cells. The proposed framework is generally useful and can be applied to different datasets acquired in a variety of laboratory settings. This is accomplished by tying together two fundamental aspects of cell lineage construction, namely cell segmentation and tracking, via a Bayesian inference of dynamic models. In contrast to most existing approaches, which aim to be general, no assumption of cell shape is made. Spatial, temporal, and cross-sectional variation of the analysed data are accommodated by two key contributions. First, time series analysis is exploited to estimate the temporal cell shape uncertainty in addition to cell trajectory. Second, a fast marching (FM) algorithm is used to integrate the inferred cell properties with the observed image measurements in order to obtain image likelihood for cell segmentation, and association. The proposed approach has been tested on eight different time-lapse microscopy data sets, some of which are high-throughput, demonstrating promising results for the detection, segmentation and association of planar cells. Our results surpass the state of the art for the Fluo-C2DL-MSC data set of the Cell Tracking Challenge (Maška et al., 2014). Copyright © 2018 Elsevier B.V. All rights reserved.

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice

PubMed Central

Lu, Shi-Jiang; Li, Feng; Yin, Hong; Feng, Qiang; Kimbrel, Erin A; Hahm, Eunsil; Thon, Jonathan N; Wang, Wei; Italiano, Joseph E; Cho, Jaehyung; Lanza, Robert

2011-01-01

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells. PMID:21221130

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

PubMed Central

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa e; Cuervo, Patricia; de Jesus, José Batista

2017-01-01

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis. PMID:28953994

Concepts in Light Microscopy of Viruses

PubMed Central

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Concepts in Light Microscopy of Viruses.

PubMed

Witte, Robert; Andriasyan, Vardan; Georgi, Fanny; Yakimovich, Artur; Greber, Urs F

2018-04-18

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Conical Refraction Bottle Beams for Entrapment of Absorbing Droplets.

PubMed

Esseling, Michael; Alpmann, Christina; Schnelle, Jens; Meissner, Robert; Denz, Cornelia

2018-03-22

Conical refraction (CR) optical bottle beams for photophoretic trapping of airborne absorbing droplets are introduced and experimentally demonstrated. CR describes the circular split-up of unpolarised light propagating along an optical axis in a biaxial crystal. The diverging and converging cones lend themselves to the construction of optical bottle beams with flexible entry points. The interaction of single inkjet droplets with an open or partly open bottle beam is shown implementing high-speed video microscopy in a dual-view configuration. Perpendicular image planes are visualized on a single camera chip to characterize the integral three-dimensional movement dynamics of droplets. We demonstrate how a partly opened optical bottle transversely confines liquid objects. Furthermore we observe and analyse transverse oscillations of absorbing droplets as they hit the inner walls and simultaneously measure both transverse and axial velocity components.

Gold leaf counter electrodes for dye-sensitized solar cells

NASA Astrophysics Data System (ADS)

Shimada, Kazuhiro; Toyoda, Takeshi

2018-03-01

In this study, a gold leaf 100 nm thin film is used as the counter electrode in dye-sensitized solar cells. The traditional method of hammering gold foil to obtain a thin gold leaf, which requires only small amounts of gold, was employed. The gold leaf was then attached to the substrate using an adhesive to produce the gold electrode. The proposed approach for fabricating counter electrodes is demonstrated to be facile and cost-effective, as opposed to existing techniques. Compared with electrodes prepared with gold foil and sputtered gold, the gold leaf counter electrode demonstrates higher catalytic activity with a cobalt-complex electrolyte and higher cell efficiency. The origin of the improved performance was investigated by surface morphology examination (scanning electron microscopy), various electrochemical analyses (cyclic voltammetry, linear sweep voltammetry, and electrochemical impedance spectroscopy), and crystalline analysis (X-ray diffractometry).

Axenic isolation of viable Giardia muris trophozoites.

PubMed

Tillotson, K D; Buret, A; Olson, M E

1991-06-01

Large numbers of viable Giardia muris trophozoites were isolated from the duodenum of experimentally infected mice 6 days after inoculation with 1,000 G. muris cysts. A series of shaking, incubation, and washing steps in the presence of the broad-spectrum antibiotic piperacillin readily provided 4.9 +/- 1.5 x 10(5) G. muris trophozoites per mouse, free of detectable contaminant organisms. Anaerobic and microaerophilic culturing and scanning electron microscopy demonstrated axenic status and high purity of the isolates. The viability of trophozoites was 98 +/- 2%. Application of this technique should permit novel immunological and epidemiological analyses of G. muris infection and biochemical investigations of this protozoan parasite.

Imaging the developing heart: synchronized time-lapse microscopy during developmental changes

NASA Astrophysics Data System (ADS)

Nelson, Carl J.; Buckley, Charlotte; Mullins, John J.; Denvir, Martin A.; Taylor, Jonathan

2018-02-01

How do you use imaging to analyse the development of the heart, which not only changes shape but also undergoes constant, high-speed, quasi-periodic changes? We have integrated ideas from prospective and retrospective optical gating to capture long-term, phase-locked developmental time-lapse videos. In this paper we demonstrate the success of this approach over a key developmental time period: heart looping, where large changes in heart shape prevent previous prospective gating approaches from capturing phase- locked videos. We use the comparison with other approaches to in vivo heart imaging to highlight the importance of collecting the most appropriate data for the biological question.

A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton.

PubMed

Nunez-Iglesias, Juan; Blanch, Adam J; Looker, Oliver; Dixon, Matthew W; Tilley, Leann

2018-01-01

We present Skan (Skeleton analysis), a Python library for the analysis of the skeleton structures of objects. It was inspired by the "analyse skeletons" plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API) allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan's measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM)-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions.

Systematic analyses of vibration noise of a vibration isolation system for high-resolution scanning tunneling microscopes.

PubMed

Iwaya, Katsuya; Shimizu, Ryota; Hashizume, Tomihiro; Hitosugi, Taro

2011-08-01

We designed and constructed an effective vibration isolation system for stable scanning tunneling microscopy measurements using a separate foundation and two vibration isolation stages (i.e., a combination of passive and active vibration isolation dampers). Systematic analyses of vibration data along the horizontal and vertical directions are present, including the vibration transfer functions of each stage and the overall vibration isolation system. To demonstrate the performance of the system, tunneling current noise measurements are conducted with and without the vibration isolation. Combining passive and active vibration isolation dampers successfully removes most of the vibration noise in the tunneling current up to 100 Hz. These comprehensive vibration noise data, along with details of the entire system, can be used to establish a clear guideline for building an effective vibration isolation system for various scanning probe microscopes and electron microscopes.

A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton

PubMed Central

Looker, Oliver; Dixon, Matthew W.; Tilley, Leann

2018-01-01

We present Skan (Skeleton analysis), a Python library for the analysis of the skeleton structures of objects. It was inspired by the “analyse skeletons” plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API) allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan’s measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM)-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions. PMID:29472997

Insights into an adipocyte whitening program

PubMed Central

Hill, Bradford G

2015-01-01

White adipose tissue plays a critical role in regulating systemic metabolism and can remodel rapidly in response to changes in nutrient availability. Nevertheless, little is known regarding the metabolic changes occurring in adipocytes during obesity. Our laboratory recently addressed this issue in a commonly used, high-fat-diet mouse model of obesity. We found remarkable changes in adipocyte metabolism that occur prior to infiltration of macrophages in expanding adipose tissue. Results of metabolomic analyses, adipose tissue respirometry, electron microscopy, and expression analyses of key genes and proteins revealed dysregulation of several metabolic pathways, loss of mitochondrial biogenetic capacity, and apparent activation of mitochondrial autophagy which were followed in time by downregulation of numerous mitochondrial proteins important for maintaining oxidative capacity. These findings demonstrate the presence of an adipocyte whitening program that may be critical for regulating adipose tissue remodeling under conditions of chronic nutrient excess. PMID:26167407

The microscopic world: A demonstration of electron microscopy for younger students

NASA Technical Reports Server (NTRS)

Horton, Linda L.

1992-01-01

The purpose is to excite students about the importance of scientific investigation and demonstrate why they should look at things in greater detail, extending beyond superficial examination. The topics covered include: microscopy, scanning electron microscopes, high magnification, and the scientific method.

The mystery of clade X: Orciraptor gen. nov. and Viridiraptor gen. nov. are highly specialised, algivorous amoeboflagellates (Glissomonadida, Cercozoa).

PubMed

Hess, Sebastian; Melkonian, Michael

2013-09-01

In freshwater ecosystems a vast diversity of elusive protists exists that specifically feed on microalgae. Due to difficulties in isolation and long-term maintenance, most of these are still poorly known. In this study stable, bacteria-free cultures of several limnetic, algivorous amoeboflagellates were investigated by light microscopy and molecular phylogenetic analyses. All strains represent naked, biflagellate cells, either occurring as rigid flagellates or as surface-attached amoebae. They perforate cell walls of certain Zygnematophyceae and Chlorophyceae (Viridiplantae) and phagocytose algal cell contents. Time-lapse microscopy revealed the feeding behaviour, locomotional processes and life histories of the amoeboflagellates. Clear differences in cell morphology and food range specificity led to the description of two new, monotypic genera Orciraptor and Viridiraptor, which occupy similar, but distinct ecological niches in aquatic ecosystems as 'necrophytophagous' and 'parasitoid' protists, respectively. Molecular phylogenetic analyses based on 18S rDNA sequence data demonstrated that Orciraptor and Viridiraptor belonged to 'clade X' within the order Glissomonadida (Cercozoa, Rhizaria). In conclusion, we established the phenotypic identity of a clade, which until now was exclusively known from environmental sequences, and erect the new family Viridiraptoridae for 'clade X'. Its algivorous members are compared with other glissomonads and nomenclatural, methodological and ecological aspects of these novel 'raptorial' amoeboflagellates are discussed. Copyright © 2013 Elsevier GmbH. All rights reserved.

Removal of bisphenol A by adsorption mechanism using PES-SiO2 composite membranes.

PubMed

Muhamad, Mimi Suliza; Salim, Mohd Razman; Lau, Woei Jye; Hadibarata, Tony; Yusop, Zulkifli

2016-08-01

Polyethersulphone (PES) membranes blended with silicon dioxide (SiO2) nanoparticles were prepared via a dry-jet wet spinning technique for the removal of bisphenol A (BPA) by adsorption mechanism. The morphology of SiO2 nanoparticles was analysed using a transmission electron microscopy and particle size distribution was also analysed. The prepared membranes were characterized by several techniques including field emission scanning electron microscopy, Fourier transform infrared spectroscopy and water contact angle. The adsorption mechanism of membrane towards BPA was evaluated by batch experiments and kinetic model. The influence of natural organic matter (NOM) in feed water on membrane BPA removal was also studied by filtration experiments. Results showed that BPA adsorption capacity as high as 53 µg/g could be achieved by the PES membrane incorporated with 2 wt% SiO2 in which the adsorption mechanism was in accordance with the pseudo-second-order kinetic model. The intraparticles diffusion model suggested that the rate limiting factor of membrane adsorption mechanism is governed by the diffusion of BPA into the membrane pores. The presence of 10 ppm NOM has reported to negatively reduce BPA removal by 24%, as it tended to compete with BPA for membrane adsorption. This work has demonstrated that PES-SiO2 membrane has the potential to eliminate trace amount of BPA from water source containing NOM.

Influence of PCL on mechanical properties and bioactivity of ZrO2-based hybrid coatings synthesized by sol-gel dip coating technique.

PubMed

Catauro, Michelina; Bollino, Flavia; Veronesi, Paolo; Lamanna, Giuseppe

2014-06-01

The biological properties of medical implants can be enhanced through surface modifications such as to provide a firm attachment of the implant. In this study, organic-inorganic hybrid coatings have been synthesized via sol-gel dip coating. They consist of an inorganic ZrO2 matrix in which different amounts of poly(ε-caprolactone) have been entrapped to improve the mechanical properties of the films. The influence of the PCL amount on the microstructural, biological and mechanical properties of the coating has been investigated. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses have shown that the hybrids used for the coating are homogenous and totally amorphous materials; Fourier transform infrared spectroscopy (FT-IR) has demonstrated that hydrogen bonds arise between the organic and inorganic phases. SEM and atomic force microscopy (AFM) have highlighted the nanostructured nature of the film. SEM and EDS analyses, after soaking the samples in a simulated body fluid (SBF), have pointed out the apatite formation on the coating surface, which proves the bone-bonding ability of the nanocomposite bioactive films. Scratch and nano-indentation tests have shown that the coating hardness, stiffness and Young's modulus decrease in the presence of large amounts of the organic phase. Copyright © 2014. Published by Elsevier B.V.

Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

PubMed

Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

2016-09-01

Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes. Copyright © 2016 Elsevier B.V. All rights reserved.

Component analyses of urinary nanocrystallites of uric acid stone formers by combination of high-resolution transmission electron microscopy, fast Fourier transformation, energy dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy.

PubMed

Sun, Xin-Yuan; Xue, Jun-Fa; Xia, Zhi-Yue; Ouyang, Jian-Ming

2015-06-01

This study aimed to analyse the components of nanocrystallites in urines of patients with uric acid (UA) stones. X-ray diffraction (XRD), Fourier transform infrared spectroscopy, high-resolution transmission electron microscopy (HRTEM), fast Fourier transformation (FFT) of HRTEM, and energy dispersive X-ray spectroscopy (EDS) were performed to analyse the components of these nanocrystallites. XRD and FFT showed that the main component of urinary nanocrystallites was UA, which contains a small amount of calcium oxalate monohydrate and phosphates. EDS showed the characteristic absorption peaks of C, O, Ca and P. The formation of UA stones was closely related to a large number of UA nanocrystallites in urine. A combination of HRTEM, FFT, EDS and XRD analyses could be performed accurately to analyse the components of urinary nanocrystallites.

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

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

PubMed

Toda, Keisuke; Isobe, Keisuke; Namiki, Kana; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2018-04-01

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

ANALYTICAL CHEMISTRY DIVISION ANNUAL PROGRESS REPORT FOR PERIOD ENDING DECEMBER 31, 1961

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

None

1962-02-01

Research and development progress is reported on analytlcal instrumentation, dlssolver-solution analyses, special research problems, reactor projects analyses, x-ray and spectrochemical analyses, mass spectrometry, optical and electron microscopy, radiochemical analyses, nuclear analyses, inorganic preparations, organic preparations, ionic analyses, infrared spectral studies, anodization of sector coils for the Analog II Cyclotron, quality control, process analyses, and the Thermal Breeder Reactor Projects Analytical Chemistry Laboratory. (M.C.G.)

Field Ion Microscopy and Atom Probe Tomography of Metamorphic Magnetite Crystals

NASA Technical Reports Server (NTRS)

Kuhlman, K.; Martens, R. L.; Kelly, T. F.; Evans, N. D.; Miller, M. K.

2001-01-01

Magnetite has been analysed using Field Ion Microscopy (FIM) and Atom Probe Tomography (APT), highly attractive techniques for the nanoanalysis of geological materials despite the difficulties inherent in analyzing semiconducting and insulating materials. Additional information is contained in the original extended abstract.

In vivo multiphoton microscopy beyond 1 mm in the brain

NASA Astrophysics Data System (ADS)

Miller, David R.; Medina, Flor A.; Hassan, Ahmed; Perillo, Evan P.; Hagan, Kristen; Kazmi, S. M. Shams; Zemelman, Boris V.; Dunn, Andrew K.

2017-02-01

We perform high-resolution, non-invasive, in vivo deep-tissue imaging of the mouse neocortex using multiphoton microscopy with a high repetition rate optical parametric amplifier laser source tunable between λ=1,100 and 1,400 nm. We demonstrate an imaging depth of 1,200 μm in vasculature and 1,160 μm in neurons. We also demonstrate deep-tissue imaging using Indocyanine Green (ICG), which is FDA approved and a promising route to translate multiphoton microscopy to human applications.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

HANFORD WASTE MINERALOGY REFERENCE REPORT

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

DISSELKAMP RS

2010-06-29

This report lists the observed mineral phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports that used experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases observed in Hanford waste.

HANFORD WASTE MINEROLOGY REFERENCE REPORT

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

DISSELKAMP RS

2010-06-18

This report lists the observed mineral phase phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports using experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases present observed in Hanford waste.

Scanning electron microscopy analysis of corrosion degradation on tinplate substrates.

PubMed

Zumelzu, E; Cabezas, C; Vera, A

2003-01-01

The degradation of electrolytic tinplate used in food containers was analysed and evaluated, using scanning electron microscopy and electrochemical measurements of microcorrosion and ion dissolution by atomic absorption to prevent food contamination caused by metal traces and to increase the durability of such tinplates.

Electron microscopy analyses and electrical properties of the layered Bi{sub 2}WO{sub 6} phase

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

Taoufyq, A.; Laboratoire Matériaux et Environnement LME, Faculté des Sciences, Université Ibn Zohr, BP 8106, Cité Dakhla, Agadir, Maroc; Département d‘Études des Réacteurs, Laboratoire Dosimétrie Capteurs Instrumentation, CEA Cadarache

2013-07-15

The bismuth tungstate Bi{sub 2}WO{sub 6} was synthesized using a classical coprecipitation method followed by a calcination process at different temperatures. The samples were characterized by X-ray diffraction, simultaneous thermogravimetry and differential thermal analysis (TGA/DTA), scanning and transmission electron microscopy (SEM, TEM) analyses. The Rietveld analysis and electron diffraction clearly confirmed the Pca2{sub 1} non centrosymmetric space group previously proposed for this phase. The layers Bi{sub 2}O{sub 2}{sup 2+} and WO{sub 4}{sup 2−} have been directly evidenced from the HRTEM images. The electrical properties of Bi{sub 2}WO{sub 6} compacted pellets systems were determined from electrical impedance spectrometry (EIS) and directmore » current (DC) analyses, under air and argon, between 350 and 700 °C. The direct current analyses showed that the conduction observed from EIS analyses was mainly ionic in this temperature range, with a small electronic contribution. Electrical change above the transition temperature of 660 °C is observed under air and argon atmospheres. The strong conductivity increase observed under argon is interpreted in terms of formation of additional oxygen vacancies coupled with electron conduction. - Graphical abstract: High resolution transmission electron microscopy: inverse fast Fourier transform giving the layered structure of the Bi{sub 2}WO{sub 6} phase, with a representation of the cell dimensions (b and c vectors). The Bi{sub 2}O{sub 2}{sup 2+} and WO{sub 4}{sup 2−} sandwiches are visible in the IFFT image. - Highlights: • Using transmission electron microscopy, we visualize the layered structure of Bi{sub 2}WO{sub 6}. • Electrical analyses under argon gas show some increase in conductivity. • The phase transition at 660 °C is evidenced from electrical modification.« less

Electron Microscopy and Analytical X-ray Characterization of Compositional and Nanoscale Structural Changes in Fossil Bone

NASA Astrophysics Data System (ADS)

Boatman, Elizabeth Marie

The nanoscale structure of compact bone contains several features that are direct indicators of bulk tissue mechanical properties. Fossil bone tissues represent unique opportunities to understand the compact bone structure/property relationships from a deep time perspective, offering a possible array of new insights into bone diseases, biomimicry of composite materials, and basic knowledge of bioapatite composition and nanoscale bone structure. To date, most work with fossil bone has employed microscale techniques and has counter-indicated the survival of bioapatite and other nanoscale structural features. The obvious disconnect between the use of microscale techniques and the discernment of nanoscale structure has prompted this work. The goal of this study was to characterize the nanoscale constituents of fossil compact bone by applying a suite of diffraction, microscopy, and spectrometry techniques, representing the highest levels of spatial and energy resolution available today, and capable of complementary structural and compositional characterization from the micro- to the nanoscale. Fossil dinosaur and crocodile long bone specimens, as well as modern ratite and crocodile femurs, were acquired from the UC Museum of Paleontology. Preserved physiological features of significance were documented with scanning electron microscopy back-scattered imaging. Electron microprobe wavelength-dispersive X-ray spectroscopy (WDS) revealed fossil bone compositions enriched in fluorine with a complementary loss of oxygen. X-ray diffraction analyses demonstrated that all specimens were composed of apatite. Transmission electron microscopy (TEM) imaging revealed preserved nanocrystallinity in the fossil bones and electron diffraction studies further identified these nanocrystallites as apatite. Tomographic analyses of nanoscale elements imaged by TEM and small angle X-ray scattering were performed, with the results of each analysis further indicating that nanoscale structure is highly conserved in these four fossil specimens. Finally, the results of this study indicate that bioapatite can be preserved in even the most ancient vertebrate specimens, further supporting the idea that fossilization is a preservational process. This work also underlines the importance of using appropriately selected characterization and analytical techniques for the study of fossil bone, especially from the perspective of spatial resolution and the scale of the bone structural features in question.

Confocal microscopy as a useful approach to describe gill rakers of Asian species of carp and native filter-feeding fishes of the upper Mississippi River system

USGS Publications Warehouse

Liza R. Walleser,; D.R. Howard,; Sandheinrich, Mark B.; Gaikowski, Mark P.; Amberg, Jon J.

2014-01-01

To better understand potential diet overlap among exotic Asian species of carp and native species of filter-feeding fishes of the upper Mississippi River system, microscopy was used to document morphological differences in the gill rakers. Analysing samples first with light microscopy and subsequently with confocal microscopy, the three-dimensional structure of gill rakers in Hypophthalmichthys molitrix,Hypophthalmichthys nobilis and Dorosoma cepedianum was more thoroughly described and illustrated than previous work with traditional microscopy techniques. The three-dimensional structure of gill rakers in Ictiobus cyprinellus was described and illustrated for the first time.

Oscheius wisconsinensis n. sp. (Nematoda: Rhabditidae), a potential entomopathogenic nematode from the marshlands of Wisconsin

USDA-ARS?s Scientific Manuscript database

Oscheius wisconsinensis n. sp. (Rhabditidae) was recovered through the Galleria bait method from a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit...

The Developmental Regulator SEEDSTICK Controls Structural and Mechanical Properties of the Arabidopsis Seed Coat

PubMed Central

Beauzamy, Léna; Caporali, Elisabetta; Koroney, Abdoul-Salam

2016-01-01

Although many transcription factors involved in cell wall morphogenesis have been identified and studied, it is still unknown how genetic and molecular regulation of cell wall biosynthesis is integrated into developmental programs. We demonstrate by molecular genetic studies that SEEDSTICK (STK), a transcription factor controlling ovule and seed integument identity, directly regulates PMEI6 and other genes involved in the biogenesis of the cellulose-pectin matrix of the cell wall. Based on atomic force microscopy, immunocytochemistry, and chemical analyses, we propose that structural modifications of the cell wall matrix in the stk mutant contribute to defects in mucilage release and seed germination under water-stress conditions. Our studies reveal a molecular network controlled by STK that regulates cell wall properties of the seed coat, demonstrating that developmental regulators controlling organ identity also coordinate specific aspects of cell wall characteristics. PMID:27624758

Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

PubMed Central

Trojanowski, Damian; Ginda, Katarzyna; Pióro, Monika; Hołówka, Joanna; Skut, Partycja; Jakimowicz, Dagmara

2015-01-01

ABSTRACT It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. PMID:25691599

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

PubMed

Havrdova, M; Polakova, K; Skopalik, J; Vujtek, M; Mokdad, A; Homolkova, M; Tucek, J; Nebesarova, J; Zboril, R

2014-12-01

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed Central

2012-01-01

Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519–17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types. PMID:22967319

Histophilus somni causes extracellular trap formation by bovine neutrophils and macrophages.

PubMed

Hellenbrand, Katrina M; Forsythe, Katelyn M; Rivera-Rivas, Jose J; Czuprynski, Charles J; Aulik, Nicole A

2013-01-01

Histophilus somni (formerly Haemophilus somnus) is a Gram-negative pleomorphic coccobacillus that causes respiratory, reproductive, cardiac and neuronal diseases in cattle. H. somni is a member of the bovine respiratory disease complex that causes severe bronchopneumonia in cattle. Previously, it has been reported that bovine neutrophils and macrophages have limited ability to phagocytose and kill H. somni. Recently, it was discovered that bovine neutrophils and macrophages produce extracellular traps in response to Mannheimia haemolytica, another member of the bovine respiratory disease complex. In this study, we demonstrate that H. somni also causes extracellular trap production by bovine neutrophils in a dose- and time-dependent manner, which did not coincide with the release of lactate dehydrogenase, a marker for necrosis. Neutrophil extracellular traps were produced in response to outer membrane vesicles, but not lipooligosacchride alone. Using scanning electron microscopy and confocal microscopy, we observed H. somni cells trapped within a web-like structure. Further analyses demonstrated that bovine neutrophils trapped and killed H. somni in a DNA-dependent manner. Treatment of DNA extracellular traps with DNase I freed H. somni cells and diminished bacterial death. Treatment of bovine monocyte-derived macrophages with H. somni cells also caused macrophage extracellular trap formation. These findings suggest that extracellular traps may play a role in the host response to H. somni infection in cattle. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles

PubMed Central

Gondikas, Andreas; von der Kammer, Frank; Hofmann, Thilo; Marchetti-Deschmann, Martina; Allmaier, Günter; Marko-Varga, György; Andersson, Roland

2017-01-01

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability. PMID:27639623

Surface topography and ordering-variant segregation in GaInP[sub 2

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

Friedman, D.J.; Zhu, J.G.; Kibbler, A.E.

1993-09-27

Using transmission electron diffraction dark-field imaging, atomic force microscopy (AFM), and Nomarski microscopy, we demonstrate a direct connection between surface topography and cation site ordering in GaInP[sub 2]. We study epilayers grown by organometallic vapor-phase epitaxy on GaAs substrates oriented 2[degree] off (100) towards (110). Nomarski microscopy shows that, as growth proceeds, the surface of ordered material forms faceted structures aligned roughly along [011]. A comparison with the dark-field demonstrates that the [1[bar 1]1] and [11[bar 1

Sputtered Pd as hydrogen storage for a chip-integrated microenergy system.

PubMed

Slavcheva, E; Ganske, G; Schnakenberg, U

2014-01-01

The work presents a research on preparation and physical and electrochemical characterisation of dc magnetron sputtered Pd films envisaged for application as hydrogen storage in a chip-integrated hydrogen microenergy system. The influence of the changes in the sputtering pressure on the surface structure, morphology, and roughness was analysed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AMF). The electrochemical activity towards hydrogen adsorption/desorption and formation of PdH were investigated in 0.5 M H2SO4 using the methods of cyclic voltammetry and galvanostatic polarisation. The changes in the electrical properties of the films as a function of the sputtering pressure and the level of hydrogenation were evaluated before and immediately after the electrochemical charging tests, using a four-probe technique. The research resulted in establishment of optimal sputter regime, ensuring fully reproducible Pd layers with highly developed surface, moderate porosity, and mechanical stability. Selected samples were integrated as hydrogen storage in a newly developed unitized microenergy system and tested in charging (water electrolysis) and discharging (fuel cell) operative mode at ambient conditions demonstrating a stable recycling performance.

Kenaf bast cellulosic fibers hierarchy: a comprehensive approach from micro to nano.

PubMed

Karimi, Samaneh; Tahir, Paridah Md; Karimi, Ali; Dufresne, Alain; Abdulkhani, Ali

2014-01-30

Cellulosic fibers from kenaf bast were isolated in three distinct stages. Initially raw kenaf bast fibers were subjected to an alkali pulping process. Then pulped fibers undergone a bleaching process and finally both pulped and bleached fibers were separated into their constituent nanoscale cellulosic fibers by mechanical shearing. The influence of each treatment on the chemical composition of fibers was investigated. Moreover morphology, functional groups, crystallinity, and thermal behavior of fiber hierarchy at different stages of purification were studied using scanning and transmission electron microscopies, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA), respectively. Microscopy studies revealed that applied procedures successfully isolated nanoscale cellulosic fibers from both unbleached and bleached pulps. Chemical composition analysis and FTIR spectroscopy showed that lignin and hemicellulose were almost entirely removed by the applied treatments. XRD and TGA analyses demonstrated progressive enhancement of properties in fibers, hierarchically, in going from micro to nano scale. Interestingly no significant evolution was observed between obtained data of characterized ubnleached and bleached nanofibers. Copyright © 2013 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.

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.

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

Synthesis and in vitro antifungal efficacy of oleoyl-chitosan nanoparticles against plant pathogenic fungi.

PubMed

Xing, Ke; Shen, Xiaoqiang; Zhu, Xiao; Ju, Xiuyun; Miao, Xiangmin; Tian, Jun; Feng, Zhaozhong; Peng, Xue; Jiang, Jihong; Qin, Sheng

2016-01-01

An antifungal dispersion system was prepared by oleoyl-chitosan (O-chitosan) nanoparticles, and the antifungal activity against several plant pathogenic fungi was investigated. Under scanning electron microscopy, the nanoparticles formulation appeared to be uniform with almost spherical shape. The particle size of nanoparticles was around 296.962 nm. Transmission electron microscopy observation showed that nanoparticles could be well distributed in potato dextrose agar medium. Mycelium growth experiment demonstrated that Nigrospora sphaerica, Botryosphaeria dothidea, Nigrospora oryzae and Alternaria tenuissima were chitosan-sensitive, while Gibberella zeae and Fusarium culmorum were chitosan-resistant. The antifungal index was increased as the concentration of nanoparticles increased for chitosan-sensitive fungi. Fatty acid analyses revealed that plasma membranes of chitosan-sensitive fungi were shown to have lower levels of unsaturated fatty acid than chitosan-resistant fungi. Phylogenetic analysis based on ITS gene sequences indicated that two chitosan-resistant fungi had a near phylogenetic relationship. Results showed that O-chitosan nanoparticles could be a useful alternative for controlling pathogenic fungi in agriculture. Copyright © 2015 Elsevier B.V. All rights reserved.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

PubMed Central

Aguet, François; Upadhyayula, Srigokul; Gaudin, Raphaël; Chou, Yi-ying; Cocucci, Emanuele; He, Kangmin; Chen, Bi-Chang; Mosaliganti, Kishore; Pasham, Mithun; Skillern, Wesley; Legant, Wesley R.; Liu, Tsung-Li; Findlay, Greg; Marino, Eric; Danuser, Gaudenz; Megason, Sean; Betzig, Eric; Kirchhausen, Tom

2016-01-01

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies. PMID:27535432

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging.

PubMed

Managò, Stefano; Migliaccio, Nunzia; Terracciano, Monica; Napolitano, Michela; Martucci, Nicola M; De Stefano, Luca; Rendina, Ivo; De Luca, Anna Chiara; Lamberti, Annalisa; Rea, Ilaria

2018-04-01

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimization for extracellular biosynthesis of silver nanoparticles by Penicillium aculeatum Su1 and their antimicrobial activity and cytotoxic effect compared with silver ions.

PubMed

Ma, Liang; Su, Wei; Liu, Jian-Xin; Zeng, Xiao-Xi; Huang, Zhi; Li, Wen; Liu, Zheng-Chun; Tang, Jian-Xin

2017-08-01

The present study addresses an eco-friendly and energy-saving method for extracellular biosynthesis of silver nanoparticles (AgNPs) using a cell free filtrate of the fungus strain Penicillium aculeatum Su1 as a reducing agent. Parametric optimization of the biosynthesis process demonstrated different effects on the size, distribution, yield, and synthesis rate of biosynthesized AgNPs. The transmission electron microscopy (TEM) measurements demonstrated that AgNPs were spherical or approximately spherical, with a size between 4 and 55nm. High-resolution transmission electron microscopy (HR-TEM) and X-ray diffraction (XRD) analyses indicated that AgNPs were nanocrystalline by nature, with the character of a face-centered cubic (fcc). Fourier transform infrared spectroscopy (FTIR) analysis confirmed the existence of protein molecules that acted as a reducing agent and a capping agent during the biosynthesis process. Furthermore, the biosynthesized AgNPs exhibited higher antimicrobial activity than silver ions against Gram negative bacteria, Gram positive bacteria and fungi. Compared with silver ions, the biosynthesized AgNPs presented higher biocompatibility toward human bronchial epithelial (HBE) cells and high cytotoxicity in a dose-dependent manner with an IC 50 of 48.73μg/mL toward A549 cells. These results demonstrate that Penicillium aculeatum Su1 is a potential bioresource that can be used to produce low-cost and eco-friendly AgNPs as efficient antimicrobial agent, drug delivery vehicle or anticancer drug for clinic treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

PubMed

Kyoung, Minjoung; Russell, Sarah J; Kohnhorst, Casey L; Esemoto, Nopondo N; An, Songon

2015-01-27

Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.

Optimizing use of the structural chemical analyser (variable pressure FESEM-EDX Raman spectroscopy) on micro-size complex historical paintings characterization.

PubMed

Guerra, I; Cardell, C

2015-10-01

The novel Structural Chemical Analyser (hyphenated Raman spectroscopy and scanning electron microscopy equipped with an X-ray detector) is gaining popularity since it allows 3-D morphological studies and elemental, molecular, structural and electronic analyses of a single complex micro-sized sample without transfer between instruments. However, its full potential remains unexploited in painting heritage where simultaneous identification of inorganic and organic materials in paintings is critically yet unresolved. Despite benefits and drawbacks shown in literature, new challenges have to be faced analysing multifaceted paint specimens. SEM-Structural Chemical Analyser systems differ since they are fabricated ad hoc by request. As configuration influences the procedure to optimize analyses, likewise analytical protocols have to be designed ad hoc. This paper deals with the optimization of the analytical procedure of a Variable Pressure Field Emission scanning electron microscopy equipped with an X-ray detector Raman spectroscopy system to analyse historical paint samples. We address essential parameters, technical challenges and limitations raised from analysing paint stratigraphies, archaeological samples and loose pigments. We show that accurate data interpretation requires comprehensive knowledge of factors affecting Raman spectra. We tackled: (i) the in-FESEM-Raman spectroscopy analytical sequence, (ii) correlations between FESEM and Structural Chemical Analyser/laser analytical position, (iii) Raman signal intensity under different VP-FESEM vacuum modes, (iv) carbon deposition on samples under FESEM low-vacuum mode, (v) crystal nature and morphology, (vi) depth of focus and (vii) surface-enhanced Raman scattering effect. We recommend careful planning of analysis strategies prior to research which, although time consuming, guarantees reliable results. The ultimate goal of this paper is to help to guide future users of a FESEM-Structural Chemical Analyser system in order to increase applications. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Fabrication of lateral lattice-polarity-inverted GaN heterostructure

NASA Astrophysics Data System (ADS)

Katayama, Ryuji; Kuge, Yoshihiro; Kondo, Takashi; Onabe, Kentaro

2007-04-01

Fabrication of the lateral polarity-inverted GaN heterostructure on sapphire (0 0 0 1) using a radio-frequency plasma enhanced molecular beam epitaxy is demonstrated. Its microscopic properties, which are closely related to the local polarity distribution, such as surface potentials, piezoelectric polarizations and residual carrier concentrations were investigated by Kelvin force microscopy and micro-Raman scattering. The successful inversion from Ga-polarity to N-polarity of GaN in a specific domain and its higher crystal perfection had been confirmed clearly by these microscopic analyses. The results were also fairly consistent with that of KOH etching experiments, which suggest the applicability of these processes to the fabrication of photonic nanostructures composed of nitride semiconductors.

Plasma-assisted reduction of silver ions impregnated into a natural zeolite framework

NASA Astrophysics Data System (ADS)

Osonio, Airah P.; Vasquez, Magdaleno R.

2018-02-01

A green, dry, and energy-efficient method for the fabrication of silver-zeolite (AgZ) composite via 13.56 MHz radio-frequency plasma reduction is demonstrated. Impregnation by soaking and ion-exchange deposition were performed to load the silver ions (Ag+) into the sodium-zeolite samples. Characterization was performed by optical emission spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, and Brunauer-Emmett-Teller analyses. Results indicate the successful reduction of Ag+ to its metallic state on the surface of the zeolite with a mean diameter of 165 nm. This plasma-induced reduction technique opens possibilities in several areas including catalysis, adsorption, water treatment, and medicine.

Wide field fluorescence epi-microscopy behind a scattering medium enabled by speckle correlations

NASA Astrophysics Data System (ADS)

Hofer, Matthias; Soeller, Christian; Brasselet, Sophie; Bertolotti, Jacopo

2018-04-01

Fluorescence microscopy is widely used in biological imaging, however scattering from tissues strongly limits its applicability to a shallow depth. In this work we adapt a methodology inspired from stellar speckle interferometry, and exploit the optical memory effect to enable fluorescence microscopy through a turbid layer. We demonstrate efficient reconstruction of micrometer-size fluorescent objects behind a scattering medium in epi-microscopy, and study the specificities of this imaging modality (magnification, field of view, resolution) as compared to traditional microscopy. Using a modified phase retrieval algorithm to reconstruct fluorescent objects from speckle images, we demonstrate robust reconstructions even in relatively low signal to noise conditions. This modality is particularly appropriate for imaging in biological media, which are known to exhibit relatively large optical memory ranges compatible with tens of micrometers size field of views, and large spectral bandwidths compatible with emission fluorescence spectra of tens of nanometers widths.

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

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.

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

Characterisation of Oscheius onirici (Nematoda: Rhabditidae), a hermaphroditic nematode from the marshlands of Wisconsin

USDA-ARS?s Scientific Manuscript database

An Oscheius (Rhabditidae) was recovered through the Galleria bait method from a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit rDNA gene (SSU), D...

Comparative study of three Plumbago L. species (Plumbaginaceae) by microscopy, UPLC–UV and HPTLC analyses

USDA-ARS?s Scientific Manuscript database

This paper presents a comparative study of anatomy of leaves, stems and roots of three species of Plumbago, namely P. auriculata Lam., P. indica L. and P. zeylanica L. by light microscopy. The paper also provides qualitative and quantitative analysis of the naphthoquinone, plumbagin, a major constit...

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

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

PubMed

Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

2016-06-06

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

A multi-emitter fitting algorithm for potential live cell super-resolution imaging over a wide range of molecular densities.

PubMed

Takeshima, T; Takahashi, T; Yamashita, J; Okada, Y; Watanabe, S

2018-05-25

Multi-emitter fitting algorithms have been developed to improve the temporal resolution of single-molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm -2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high-density super-resolution microscopy), at densities up to 20 molecules μm -2 , and better than DAOSTORM at higher molecular densities. The application of WTM to a high-density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high-density reconstruction. WTM algorithm is a computationally fast, multi-emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

PubMed

Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

2018-01-01

Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

PubMed Central

Knecht, Hans; Dobrucki, Jurek; Mai, Sabine

2018-01-01

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine. PMID:29297245

Nuclear microscopy of sperm cell elemental structure

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

Bench, G.S.; Balhorn, R.; Friz, A.M.

1994-09-28

Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses.more » Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.« less

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

Evaluation of the Roche LightMix Gastro parasites multiplex PCR assay detecting Giardia duodenalis, Entamoeba histolytica, cryptosporidia, Dientamoeba fragilis, and Blastocystis hominis.

PubMed

Friesen, J; Fuhrmann, J; Kietzmann, H; Tannich, E; Müller, M; Ignatius, R

2018-03-23

Multiplex PCR assays offer highly sensitive and specific tools for the detection of enteric pathogens. This prospective study aimed at comparing the novel Roche LightMix Modular Assay Gastro Parasites (LMAGP) detecting Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Blastocystishominis, and Dientamoebafragilis with routine laboratory procedures. Stool specimens (n = 1062 from 1009 patients) were consecutively examined by LMAGP, R-Biopharm Ridascreen enzyme immunoassays (EIAs) detecting G. duodenalis or E. histolytica/dispar, and microscopy of wet mounts. Discrepant results were analysed by in-house PCR. D. fragilis or B. hominis were detected by LMAGP in 131 (14.4%) and 179 (19.9%; 16 samples positive by microscopy; p < 0.0001) of 909 samples, respectively. Of 918 samples analysed for Cryptosporidium spp., six were positive by LMAGP (three could be confirmed by Kinyoun staining and one by in-house PCR). G. duodenalis was detected by LMAGP, EIA, or microscopy in 20, 16, or 9 of 1039 stool samples, respectively; all four samples missed by EIA were confirmed by in-house PCR. In total, 938 stool samples were analysed for E. histolytica/dispar. Nine of ten EIA-positive samples were negative by LMAGP but positive by in-house PCR for E. dispar. One E. histolytica infection (positive by both LMAGP and in-house PCR) was missed by EIA and microscopy. Parasites only detected by microscopy included Enterobius vermicularis eggs (n = 3) and apathogenic amoebae (n = 27). The data call for routine use of multiplex PCR assays for the detection of enteric protozoan parasites in laboratory diagnostics. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Nanometer-scale characterization of exceptionally preserved bacterial fossils in Paleocene phosphorites from Ouled Abdoun (Morocco).

PubMed

Cosmidis, J; Benzerara, K; Gheerbrant, E; Estève, I; Bouya, B; Amaghzaz, M

2013-03-01

Micrometer-sized spherical and rod-shaped forms have been reported in many phosphorites and often interpreted as microbes fossilized by apatite, based on their morphologic resemblance with modern bacteria inferred by scanning electron microscopy (SEM) observations. This interpretation supports models involving bacteria in the formation of phosphorites. Here, we studied a phosphatic coprolite of Paleocene age originating from the Ouled Abdoun phosphate basin (Morocco) down to the nanometer-scale using focused ion beam milling, transmission electron microscopy (TEM), and scanning transmission x-ray microscopy (STXM) coupled with x-ray absorption near-edge structure spectroscopy (XANES). The coprolite, exclusively composed of francolite (a carbonate-fluroapatite), is formed by the accumulation of spherical objects, delimited by a thin envelope, and whose apparent diameters are between 0.5 and 3 μm. The envelope of the spheres is composed of a continuous crown dense to electrons, which measures 20-40 nm in thickness. It is surrounded by two thinner layers that are more porous and transparent to electrons and enriched in organic carbon. The observed spherical objects are very similar with bacteria encrusting in hydroxyapatite as observed in laboratory experiments. We suggest that they are Gram-negative bacteria fossilized by francolite, the precipitation of which started within the periplasm of the cells. We discuss the role of bacteria in the fossilization mechanism and propose that they could have played an active role in the formation of francolite. This study shows that ancient phosphorites can contain fossil biological subcellular structures as fine as a bacterial periplasm. Moreover, we demonstrate that while morphological information provided by SEM analyses is valuable, the use of additional nanoscale analyses is a powerful approach to help inferring the biogenicity of biomorphs found in phosphorites. A more systematic use of this approach could considerably improve our knowledge and understanding of the microfossils present in the geological record. © 2012 Blackwell Publishing Ltd.

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

NASA Astrophysics Data System (ADS)

Spinato, Cinzia; Perez Ruiz de Garibay, Aritz; Kierkowicz, Magdalena; Pach, Elzbieta; Martincic, Markus; Klippstein, Rebecca; Bourgognon, Maxime; Wang, Julie Tzu-Wen; Ménard-Moyon, Cécilia; Al-Jamal, Khuloud T.; Ballesteros, Belén; Tobias, Gerard; Bianco, Alberto

2016-06-01

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells.In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07923c

Development of a fibre size-specific job-exposure matrix for airborne asbestos fibres.

PubMed

Dement, J M; Kuempel, E D; Zumwalde, R D; Smith, R J; Stayner, L T; Loomis, D

2008-09-01

To develop a method for estimating fibre size-specific exposures to airborne asbestos dust for use in epidemiological investigations of exposure-response relations. Archived membrane filter samples collected at a Charleston, South Carolina asbestos textile plant during 1964-8 were analysed by transmission electron microscopy (TEM) to determine the bivariate diameter/length distribution of airborne fibres by plant operation. The protocol used for these analyses was based on the direct transfer method published by the International Standards Organization (ISO), modified to enhance fibre size determinations, especially for long fibres. Procedures to adjust standard phase contrast microscopy (PCM) fibre concentration measures using the TEM data in a job-exposure matrix (JEM) were developed in order to estimate fibre size-specific exposures. A total of 84 airborne dust samples were used to measure diameter and length for over 18,000 fibres or fibre bundles. Consistent with previous studies, a small proportion of airborne fibres were longer than >5 microm in length, but the proportion varied considerably by plant operation (range 6.9% to 20.8%). The bivariate diameter/length distribution of airborne fibres was expressed as the proportion of fibres in 20 size-specific cells and this distribution demonstrated a relatively high degree of variability by plant operation. PCM adjustment factors also varied substantially across plant operations. These data provide new information concerning the airborne fibre characteristics for a previously studied textile facility. The TEM data demonstrate that the vast majority of airborne fibres inhaled by the workers were shorter than 5 mum in length, and thus not included in the PCM-based fibre counts. The TEM data were used to develop a new fibre size-specific JEM for use in an updated cohort mortality study to investigate the role of fibre dimension in the development of asbestos-related lung diseases.

Two Simple Classroom Demonstrations for Scanning Probe Microscopy Based on a Macroscopic Analogy

ERIC Educational Resources Information Center

Hajkova, Zdenka; Fejfar, Antonin; Smejkal, Petr

2013-01-01

This article describes two simple classroom demonstrations that illustrate the principles of scanning probe microscopy (SPM) based on a macroscopic analogy. The analogy features the bumps in an egg carton to represent the atoms on a chemical surface and a probe that can be represented by a dwarf statue (illustrating an origin of the prefix…

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

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.

Mesoporous silica coatings for cephalosporin active release at the bone-implant interface

NASA Astrophysics Data System (ADS)

Rădulescu, Dragoş; Voicu, Georgeta; Oprea, Alexandra Elena; Andronescu, Ecaterina; Grumezescu, Valentina; Holban, Alina Maria; Vasile, Bogdan Stefan; Surdu, Adrian Vasile; Grumezescu, Alexandru Mihai; Socol, Gabriel; Mogoantă, Laurenţiu; Mogoşanu, George Dan; Balaure, Paul Cătălin; Rădulescu, Radu; Chifiriuc, Mariana Carmen

2016-06-01

In this study, we investigated the potential of MAPLE-deposited coatings mesoporous silica nanoparticles (MSNs) to release Zinforo (ceftarolinum fosmil) in biologically active form. The MSNs were prepared by using a classic procedure with cetyltrimethylammonium bromide as sacrificial template and tetraethylorthosilicate as the monomer. The Brunauer-Emmett-Teller (BET) and transmission electron microscopy (TEM) analyses revealed network-forming granules with diameters under 100 nm and an average pore diameter of 2.33 nm. The deposited films were characterized by SEM, TEM, XRD and IR. Microbiological analyses performed on ceftaroline-loaded films demonstrated that the antibiotic was released in an active form, decreasing the microbial adherence rate and colonization of the surface. Moreover, the in vitro and in vivo assays proved the excellent biodistribution and biocompatibility of the prepared systems. Our results suggest that the obtained bioactive coatings possess a significant potential for the design of drug delivery systems and antibacterial medical-use surfaces, with great applications in bone implantology.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

PubMed Central

Mori, Tetsuya; Saveliev, Sergei V.; Xu, Yao; Stafford, Walter F.; Cox, Michael M.; Inman, Ross B.; Johnson, Carl H.

2002-01-01

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns. PMID:12477935

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

PubMed

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Evaluation of Sidestream Darkfield Microscopy for Real-Time Imaging Acellular Dermal Matrix Revascularization.

PubMed

DeGeorge, Brent R; Olenczak, J Bryce; Cottler, Patrick S; Drake, David B; Lin, Kant Y; Morgan, Raymond F; Campbell, Christopher A

2016-06-01

Acellular dermal matrices (ADMs) serve as a regenerative framework for host cell integration and collagen deposition to augment the soft tissue envelope in ADM-assisted breast reconstruction-a process dependent on vascular ingrowth. To date noninvasive intra-operative imaging techniques have been inadequate to evaluate the revascularization of ADM. We investigated the safety, feasibility, and efficacy of sidestream darkfield (SDF) microscopy to assess the status of ADM microvascular architecture in 8 patients at the time of tissue expander to permanent implant exchange during 2-stage ADM-assisted breast reconstruction. The SDF microscopy is a handheld device, which can be used intraoperatively for the real-time assessment of ADM blood flow, vessel density, vessel size, and branching pattern. The SDF microscopy was used to assess the microvascular architecture in the center and border zone of the ADM and to compare the native, non-ADM-associated capsule in each patient as a within-subject control. No incidences of periprosthetic infection, explantation, or adverse events were reported after SDF image acquisition. Native capsules demonstrate a complex, layered architecture with an average vessel area density of 14.9 mm/mm and total vessel length density of 12.3 mm/mm. In contrast to native periprosthetic capsules, ADM-associated capsules are not uniformly vascularized structures and demonstrate 2 zones of microvascular architecture. The ADM and native capsule border zone demonstrates palisading peripheral vascular arcades with continuous antegrade flow. The central zone of the ADM demonstrates punctate perforating vascular plexi with intermittent, sluggish flow, and intervening 2- to 3-cm watershed zones. Sidestream darkfield microscopy allows for real-time intraoperative assessment of ADM revascularization and serves as a potential methodology to compare revascularization parameters among commercially available ADMs. Thr SDF microscopy demonstrates that the periprosthetic capsule in ADM-assisted implant-based breast reconstruction is not a uniformly vascularized structure.

Developing standards for malaria microscopy: external competency assessment for malaria microscopists in the Asia-Pacific.

PubMed

Ashraf, Sania; Kao, Angie; Hugo, Cecilia; Christophel, Eva M; Fatunmbi, Bayo; Luchavez, Jennifer; Lilley, Ken; Bell, David

2012-10-24

Malaria diagnosis has received renewed interest in recent years, associated with the increasing accessibility of accurate diagnosis through the introduction of rapid diagnostic tests and new World Health Organization guidelines recommending parasite-based diagnosis prior to anti-malarial therapy. However, light microscopy, established over 100 years ago and frequently considered the reference standard for clinical diagnosis, has been neglected in control programmes and in the malaria literature and evidence suggests field standards are commonly poor. Microscopy remains the most accessible method for parasite quantitation, for drug efficacy monitoring, and as a reference of assessing other diagnostic tools. This mismatch between quality and need highlights the importance of the establishment of reliable standards and procedures for assessing and assuring quality. This paper describes the development, function and impact of a multi-country microscopy external quality assurance network set up for this purpose in Asia. Surveys were used for key informants and past participants for feedback on the quality assurance programme. Competency scores for each country from 14 participating countries were compiled for analyses using paired sample t-tests. In-depth interviews were conducted with key informants including the programme facilitators and national level microscopists. External assessments and limited retraining through a formalized programme based on a reference slide bank has demonstrated an increase in standards of competence of senior microscopists over a relatively short period of time, at a potentially sustainable cost. The network involved in the programme now exceeds 14 countries in the Asia-Pacific, and the methods are extended to other regions. While the impact on national programmes varies, it has translated in some instances into a strengthening of national microscopy standards and offers a possibility both for supporting revival of national microcopy programmes, and for the development of globally recognized standards of competency needed both for patient management and field research.

Developing standards for malaria microscopy: external competency assessment for malaria microscopists in the Asia-Pacific

PubMed Central

2012-01-01

Background Malaria diagnosis has received renewed interest in recent years, associated with the increasing accessibility of accurate diagnosis through the introduction of rapid diagnostic tests and new World Health Organization guidelines recommending parasite-based diagnosis prior to anti-malarial therapy. However, light microscopy, established over 100 years ago and frequently considered the reference standard for clinical diagnosis, has been neglected in control programmes and in the malaria literature and evidence suggests field standards are commonly poor. Microscopy remains the most accessible method for parasite quantitation, for drug efficacy monitoring, and as a reference of assessing other diagnostic tools. This mismatch between quality and need highlights the importance of the establishment of reliable standards and procedures for assessing and assuring quality. This paper describes the development, function and impact of a multi-country microscopy external quality assurance network set up for this purpose in Asia. Methods Surveys were used for key informants and past participants for feedback on the quality assurance programme. Competency scores for each country from 14 participating countries were compiled for analyses using paired sample t-tests. In-depth interviews were conducted with key informants including the programme facilitators and national level microscopists. Results External assessments and limited retraining through a formalized programme based on a reference slide bank has demonstrated an increase in standards of competence of senior microscopists over a relatively short period of time, at a potentially sustainable cost. The network involved in the programme now exceeds 14 countries in the Asia-Pacific, and the methods are extended to other regions. Conclusions While the impact on national programmes varies, it has translated in some instances into a strengthening of national microscopy standards and offers a possibility both for supporting revival of national microcopy programmes, and for the development of globally recognized standards of competency needed both for patient management and field research. PMID:23095668

Aquaporin-0 Targets Interlocking Domains to Control the Integrity and Transparency of the Eye Lens

PubMed Central

Lo, Woo-Kuen; Biswas, Sondip K.; Brako, Lawrence; Shiels, Alan; Gu, Sumin; Jiang, Jean X.

2014-01-01

Purpose. Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. Methods. The loss of AQP0 in AQP0−/− lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0−/− lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. Results. Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. Conclusions. This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens. PMID:24458158

Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

PubMed

Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

2016-09-01

Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

TiO₂-Based Photocatalytic Geopolymers for Nitric Oxide Degradation.

PubMed

Strini, Alberto; Roviello, Giuseppina; Ricciotti, Laura; Ferone, Claudio; Messina, Francesco; Schiavi, Luca; Corsaro, Davide; Cioffi, Raffaele

2016-06-24

This study presents an experimental overview for the development of photocatalytic materials based on geopolymer binders as catalyst support matrices. Particularly, geopolymer matrices obtained from different solid precursors (fly ash and metakaolin), composite systems (siloxane-hybrid, foamed hybrid), and curing temperatures (room temperature and 60 °C) were investigated for the same photocatalyst content (i.e., 3% TiO₂ by weight of paste). The geopolymer matrices were previously designed for different applications, ranging from insulating (foam) to structural materials. The photocatalytic activity was evaluated as NO degradation in air, and the results were compared with an ordinary Portland cement reference. The studied matrices demonstrated highly variable photocatalytic performance depending on both matrix constituents and the curing temperature, with promising activity revealed by the geopolymers based on fly ash and metakaolin. Furthermore, microstructural features and titania dispersion in the matrices were assessed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analyses. Particularly, EDS analyses of sample sections indicated segregation effects of titania in the surface layer, with consequent enhancement or depletion of the catalyst concentration in the active sample region, suggesting non-negligible transport phenomena during the curing process. The described results demonstrated that geopolymer binders can be interesting catalyst support matrices for the development of photocatalytic materials and indicated a large potential for the exploitation of their peculiar features.

The role of 11-cis-retinyl esters in vertebrate cone vision.

PubMed

Babino, Darwin; Perkins, Brian D; Kindermann, Aljoscha; Oberhauser, Vitus; von Lintig, Johannes

2015-01-01

A cycle of cis-to-trans isomerization of the chromophore is intrinsic to vertebrate vision where rod and cone photoreceptors mediate dim- and bright-light vision, respectively. Daylight illumination can greatly exceed the rate at which the photoproduct can be recycled back to the chromophore by the canonical visual cycle. Thus, an additional supply pathway(s) must exist to sustain cone-dependent vision. Two-photon microscopy revealed that the eyes of the zebrafish (Danio rerio) contain high levels of 11-cis-retinyl esters (11-REs) within the retinal pigment epithelium. HPLC analyses demonstrate that 11-REs are bleached by bright light and regenerated in the dark. Pharmacologic treatment with all-trans-retinylamine (Ret-NH2), a potent and specific inhibitor of the trans-to-cis reisomerization reaction of the canonical visual cycle, impeded the regeneration of 11-REs. Intervention with 11-cis-retinol restored the regeneration of 11-REs in the presence of all-trans-Ret-NH2. We used the XOPS:mCFP transgenic zebrafish line with a functional cone-only retina to directly demonstrate that this 11-RE cycle is critical to maintain vision under bright-light conditions. Thus, our analyses reveal that a dark-generated pool of 11-REs helps to supply photoreceptors with the chromophore under the varying light conditions present in natural environments. © FASEB.

First Record of Raillietina celebensis (Cestoda: Cyclophyllidea) in South America: Redescription and Phylogeny.

PubMed

de Oliveira Simões, Raquel; Simões, Susana Balmant Enrique; Luque, José Luis; Iñiguez, Alena Mayo; Júnior, Arnaldo Maldonado

2017-08-01

Raillietina celebensis is a cestode that parasitizes the small intestine of rats and humans. Here, we detail the morphology and morphometry of R. celebensis based on specimens collected from Rattus norvegicus in the municipality of São Gonçalo, state of Rio de Janeiro, Brazil, by light and confocal scanning laser microscopies and also report the results of molecular phylogenetic analyses to determine its relationships within the family Davaineidae. Analysis of the number and size of testes, number and shape of rostellar hooks, cirrus sac length, capsules and eggs per capsule, and morphology of the mature proglottid allowed concluding that the present specimens constitute a new record of R. celebensis in South America. Our genetic and phylogenetic analyses, based on the partial small subunit 18S rRNA gene, revealed R. celebensis to be in the family Davaineidae within the genus Raillietina, in agreement with the morphological taxonomy. Phylogenetic trees obtained by neighbor-joining and maximum likelihood methods demonstrated R. celebensis as a unique taxonomic unit, and also demonstrated some taxonomic inconsistences. The incorporation of Brazilian R. celebensis sequences derived from mammals in the phylogeny of davaineids is consistent with the assertion that neither Raillietina nor Fuhrmannetta can be supported as distinct genera.

Repurposing a photosynthetic antenna protein as a super-resolution microscopy label.

PubMed

Barnett, Samuel F H; Hitchcock, Andrew; Mandal, Amit K; Vasilev, Cvetelin; Yuen, Jonathan M; Morby, James; Brindley, Amanda A; Niedzwiedzki, Dariusz M; Bryant, Donald A; Cadby, Ashley J; Holten, Dewey; Hunter, C Neil

2017-12-01

Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd 1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.

Removal of aqueous chromium and environmental CO2 by using photocatalytic TiO2 doped with tungsten.

PubMed

Trejo-Valdez, M; Hernández-Guzmán, S R; Manriquez-Ramírez, M E; Sobral, H; Martínez-Gutiérrez, H; Torres-Torres, C

2018-05-15

Removal of hexavalent chromium was accomplished by using photocatalyst materials of TiO 2 doped with tungsten oxide, environmental air as oxygen supply and white light as irradiation source. Dichromate anions in concentration ranges of 50 to 1000 μg/L were removed by means of aqueous dispersions of TiO 2 doped with tungsten. The aqueous chromium analyses were performed by Differential Pulse Voltammetry technique. Additionally, mineralization of CO 2 gas was promoted by the photocatalysis process, as was clearly shown by Raman spectroscopy and X-ray Photoelectron Spectroscopy (XPS) analyses obtained from the TiO 2 samples recovered after photocatalytic experiments. Results of sample analyses by Scanning Electron Microscopy (SEM) and High Resolution Transmission Electron Microscopy (HRTEM) are presented and discussed. Copyright © 2018 Elsevier B.V. All rights reserved.

Quietly Building Capabilities: New Instruments, Expertise, "Quiet Wing" Available at DOE User Facility

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

Lea, Alan S.; Kabius, Bernd C.; Arey, Bruce W.

2011-09-01

This feature article is prepared for publication in Microscopy Today. The goal is to communicate the value of the Quiet Wing, EMSL’s growing microscopy capability, and the science they enable to the microscopy community and hopefully various related research communities (e.g. catalysis, etc.). The secondary goals are to demonstrate EMSL’s leadership in microscopy and show our DOE client we are making excellent use of ARRA and other investments.

Electrostatically confined nanoparticle interactions and dynamics.

PubMed

Eichmann, Shannon L; Anekal, Samartha G; Bevan, Michael A

2008-02-05

We report integrated evanescent wave and video microscopy measurements of three-dimensional trajectories of 50, 100, and 250 nm gold nanoparticles electrostatically confined between parallel planar glass surfaces separated by 350 and 600 nm silica colloid spacers. Equilibrium analyses of single and ensemble particle height distributions normal to the confining walls produce net electrostatic potentials in excellent agreement with theoretical predictions. Dynamic analyses indicate lateral particle diffusion coefficients approximately 30-50% smaller than expected from predictions including the effects of the equilibrium particle distribution within the gap and multibody hydrodynamic interactions with the confining walls. Consistent analyses of equilibrium and dynamic information in each measurement do not indicate any roles for particle heating or hydrodynamic slip at the particle or wall surfaces, which would both increase diffusivities. Instead, lower than expected diffusivities are speculated to arise from electroviscous effects enhanced by the relative extent (kappaa approximately 1-3) and overlap (kappah approximately 2-4) of electrostatic double layers on the particle and wall surfaces. These results demonstrate direct, quantitative measurements and a consistent interpretation of metal nanoparticle electrostatic interactions and dynamics in a confined geometry, which provides a basis for future similar measurements involving other colloidal forces and specific biomolecular interactions.

Use of energy filtering transmission electron microscopy for image generation and element analysis in plant organisms.

PubMed

Lütz-Meindl, Ursula

2007-01-01

Energy filtering TEM (EFTEM) with modern spectrometers and software offers new possibilities for element analysis and image generation in plant cells. In the present review, applications of EFTEM in plant physiology, such as identification of light elements and ion transport, analyses of natural cell incrustations, determination of element exchange between fungi and rootlets during mycorrhiza development, heavy metal storage and detoxification, and employment in plant physiological experiments are summarized. In addition, it is demonstrated that EFTEM can be successfully used in more practical approaches, for example, in phytoremediation, food and wood industry, and agriculture. Preparation methods for plant material as prerequisites for EFTEM analysis are compared with respect to their suitability and technical problems are discussed.

Electron Spectroscopy for Chemical Analysis (ESCA) study of atmospheric particles

NASA Technical Reports Server (NTRS)

Dillard, J. G.; Seals, R. D.; Wightman, J. P.

1979-01-01

The results of analyses by ESCA (Electron Spectroscopy for Chemical Analysis) on several Nuclepore filters which were exposed during air pollution studies are presented along with correlative measurements by Neutron Activation Analysis and Scanning Electron Microscopy. Samples were exposed during air pollution studies at Norfolk, Virginia and the NASA Kennedy Space Center (KSC). It was demonstrated that with the ESCA technique it was possible to identify the chemical (bonding) state of elements contained in the atmospheric particulate matter collected on Nuclepore filters. Sulfur, nitrogen, mercury, chlorine, alkali, and alkaline earth metal species were identified in the Norfolk samples. ESCA binding energy data for aluminum indicated that three chemically different types of aluminum are present in the launch and background samples from NASA-KSC.

Low temperature synthesis of seed mediated CuO bundle of nanowires, their structural characterisation and cholesterol detection.

PubMed

Ibupoto, Z H; Khun, K; Liu, X; Willander, M

2013-10-01

In this study, we have successfully synthesised CuO bundle of nanowires using simple, cheap and low temperature hydrothermal growth method. The growth parameters such as precursor concentration and time for duration of growth were optimised. The field emission scanning electron microscopy (FESEM) has demonstrated that the CuO bundles of nanowires are highly dense, uniform and perpendicularly oriented to the substrate. The high resolution transmission electron microscopy (HRTEM) has demonstrated that the CuO nanostructures consist of bundle of nanowires and their growth pattern is along the [010] direction. The X-ray diffraction (XRD) technique described that CuO bundle of nanowires possess the monoclinic crystal phase. The surface and chemical composition analyses were carried out with X-ray photoelectron spectroscopy (XPS) technique and the obtained results suggested the pure crystal state of CuO nanostructures. In addition, the CuO nanowires were used for the cholesterol sensing application by immobilising the cholesterol oxidase through electrostatic attraction. The infrared reflection absorption spectroscopy study has also revealed that CuO nanostructures are consisting of only CuO bonding and has also shown the possible interaction of cholesterol oxidase with the sharp edge surface of CuO bundle of nanowires. The proposed cholesterol sensor has demonstrated the wide range of detection of cholesterol with good sensitivity of 33.88±0.96 mV/decade. Moreover, the CuO bundle of nanowires based sensor electrode has revealed good repeatability, reproducibility, stability, selectivity and a fast response time of less than 10s. The cholesterol sensor based on the immobilised cholesterol oxidase has good potential applicability for the determination of cholesterol from the human serum and other biological samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model.

PubMed

Diaz, José A; Hawley, Angela E; Alvarado, Christine M; Berguer, Alexandra M; Baker, Nichole K; Wrobleski, Shirley K; Wakefield, Thomas W; Lucchesi, Benedict R; Myers, Daniel D

2010-08-01

Several rodent models have been used to study deep venous thrombosis (DVT). However, a model that generates consistent venous thrombi in the presence of continuous blood flow, to evaluate therapeutic agents for DVT, is not available. Mice used in the present study were wild-type C57BL/6 (WT), plasminogen activator inhibitor-1 (PAI-1) knock out (KO) and Delta Cytoplasmic Tail (DCT). An electrolytic inferior vena cava (IVC) model (EIM) was used. A 25G stainless-steel needle, attached to a silver coated copper wire electrode (anode), was inserted into the exposed caudal IVC. Another electrode (cathode) was placed subcutaneously. A current of 250 muAmps over 15 minutes was applied. Ultrasound imaging was used to demonstrate the presence of IVC blood flow. Analyses included measurement of plasma soluble P-selectin (sP-Sel), thrombus weight (TW), vein wall morphometrics, P-selectin and Von Willebrand factor (vWF) staining, transmission electron microscopy (TEM), scanning electron microscopy (SEM); and the effect of enoxaparin on TW was evaluated. A current of 250 muAmps over 15 minutes consistently promoted thrombus formation in the IVC. Plasma sP-Sel was decreased in PAI-1 KO and increased in DCT vs. WT (WT/PAI-1: p=0.003, WT/DCT: p=0.0002). Endothelial activation was demonstrated by SEM, TEM, P-selectin and vWF immunohistochemistry and confirmed by inflammatory cell counts. Ultrasound imaging demonstrated thrombus formation in the presence of blood flow. Enoxaparin significantly reduced the thrombus size by 61% in this model. This EIM closely mimics clinical venous disease and can be used to study endothelial cell activation, leukocyte migration, thrombogenesis and therapeutic applications in the presence of blood flow.

DMD-based quantitative phase microscopy and optical diffraction tomography

NASA Astrophysics Data System (ADS)

Zhou, Renjie

2018-02-01

Digital micromirror devices (DMDs), which offer high speed and high degree of freedoms in steering light illuminations, have been increasingly applied to optical microscopy systems in recent years. Lately, we introduced DMDs into digital holography to enable new imaging modalities and break existing imaging limitations. In this paper, we will first present our progress in using DMDs for demonstrating laser-illumination Fourier ptychographic microscopy (FPM) with shotnoise limited detection. After that, we will present a novel common-path quantitative phase microscopy (QPM) system based on using a DMD. Building on those early developments, a DMD-based high speed optical diffraction tomography (ODT) system has been recently demonstrated, and the results will also be presented. This ODT system is able to achieve video-rate 3D refractive-index imaging, which can potentially enable observations of high-speed 3D sample structural changes.

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

Fluorescent light mediated a green synthesis of silver nanoparticles using the protein extract of weaver ant larvae.

PubMed

Khamhaengpol, Arunrat; Siri, Sineenat

2016-10-01

Alternative to crude plant extracts, a crude protein extract derived from animal cells is one of the potential sources of biomolecules for mediating a reduction of silver ions and a formation of silver nanoparticles (AgNPs) under a mild condition, which very few works have been reported. This work demonstrated a use of the protein extract of weaver ant larvae as a bio-facilitator for a simple, green synthesis of AgNPs under fluorescent light at room temperature. The protein extract of weaver ant larvae exhibited the reducing and antioxidant activities, which assisted a formation of AgNPs in the reaction containing only silver nitrate under light exposure. Transmission electron microscopy images revealed the dispersed, spherical AgNPs with an average size of 7.87±2.54nm. The maximum surface plasmon resonance (SPR) band of the synthesized AgNPs was at 435nm. The energy-dispersive X-ray analysis revealed that silver was a major element of the particles. The identity of AgNPs was confirmed by X-ray diffraction pattern, selected area electron diffraction and high resolution transmission electron microscopy analyses, which demonstrated the planes of face centered cubic silver. The synthesized AgNPs showed antibacterial activity against both Escherichia coli and Staphylococcus aureus with the minimum bactericidal concentration (MBC) values equally at 250μg/ml, suggesting their potential application as an effective antibacterial agent. Copyright © 2016 Elsevier B.V. All rights reserved.

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles.

PubMed

Urey, Carlos; Weiss, Victor U; Gondikas, Andreas; von der Kammer, Frank; Hofmann, Thilo; Marchetti-Deschmann, Martina; Allmaier, Günter; Marko-Varga, György; Andersson, Roland

2016-11-20

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Exploring photoinactivation of microbial biofilms using laser scanning microscopy and confined two-photon excitation.

PubMed

Thomsen, Hanna; Graf, Fabrice E; Farewell, Anne; Ericson, Marica B

2018-05-21

One pertinent complication in bacterial infection is the growth of biofilms, i.e., communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms. The effects of complexation of curcumin with hydroxypropyl-γ-cyclodextrin (HPγCD) were studied. It was shown that HPγCD-curcumin demonstrated higher bioavailability in the biofilms compared to curcumin, without affecting the subcellular uptake. Spectral quantification following photodynamic inactivation demonstrates a method for monitoring elimination of biofilms in real time using noninvasive 3D imaging. Additionally, spatially confined two-photon inactivation was demonstrated for the first time in biofilms. These results support the feasibility of advanced optical microscopy as a sensitive tool for evaluating treatment efficacy in biofilms towards improved mechanistic studies of photodynamic inactivation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Electron microscopy investigations of nanoparticles for cancer diagnostic applications

NASA Astrophysics Data System (ADS)

Koh, Ai Leen

This dissertation concerns electron microscopy characterization of magnetic (MNP) and surface enhanced Raman scattering (SERS) nanoparticles for in-vitro cancer diagnostic applications. Electron microscopy is an essential characterization tool owing to its (sub) nanometer spatial resolution. Structural information about the nanoparticles can be obtained using transmission electron microscopy (TEM), which can in turn be correlated to their physical characteristics. The scanning electron microscope (SEM) has excellent depth of field and can be effectively utilized to obtain high resolution information about nanoparticles binding onto cell surfaces. Part One of this thesis focuses on MNPs for bio-sensing and detection applications. As a preliminary study, chemically-synthesized, commercially-available iron oxide nanoparticles were compared against their laboratory-synthesized counterparts to assess their suitability for this application. The motivation for this initial study came about due to the lack of published data on commercially available iron oxide nanoparticles. TEM studies show that the latter are "beads" composed of multiple iron oxide cores encapsulated by a polymer shell, with large standard deviations in core diameter. Laboratory-synthesized iron oxide nanoparticles, on the other hand, are single core particles with small variations in diameter and therefore are expected to be better candidates for the required application. A key limitation in iron oxide nanoparticles is their relatively weak magnetic signals. The development of high moment Synthetic Anti-Ferromagnetic (SAF) nanoparticles aims to overcome this issue. SAFs are a novel class of MNPs fabricated using nanoimprint lithography, direct deposition of multilayer structure and final suspension into liquid medium (water). TEM analyses of cross-section specimens reveal that the SAFs possess characteristics similar to those of sputtered magnetic multilayer thin films. Their layered structure is preserved after a chemical etch. Magnetic measurements show a slight decrease in magnetic moment after ion milling. From TEM characterization, the introduction of oxygen into the copper release layer, prior the film deposition process, can effectively control the topography of the oxidized-copper grains and, consequently, lead to the production of SAF nanoparticles with flatter layers. Size distribution studies performed on SAFs fabricated using self-assembled stamps show that it is possible to produce monodisperse nanoparticles with diameters from 70 nm up. Part Two of the dissertation describes structural characterization experiments performed on Composite Organic-Inorganic Nanoparticles (COINs), which are a novel type of SERS nanoclusters formed by aggregating silver nanoparticles with Raman molecules, and then encapsulating them with an organic coating that stabilizes the aggregates and promotes subsequent functionalization with antibodies. Part Three of this dissertation focuses on the development and application of electron microscopy-based techniques to characterize the nanomaterial-biology interactions, to assess how, or indeed whether, nanoparticles are attaching to the cancer cells. The technique of negative staining was applied to simultaneously visualize inorganic nanoparticles and their biofunctionalized entities under the TEM and to verify the successful functionalization of nanoparticles with antibodies. The interpretation of the negatively-stained COINs was consistent with the EFTEM data. Next, the localization and characterization of CD54-functionalized COINs on the apicolateral portions of U937 leukemia cell lines was determined using TEM, SEM and Scanning Auger Microscopy. The analyses show that CD54 antigens are localized at a specific region on U937 leukemia cell surfaces. SEM imaging and SER spectroscopy correlation studies of different antibody-conjugated COINs attached onto different cancer cell lines show a direct correlation between the number of COINs binding to cells and the corresponding SER intensity. Finally, TEM was used to locate intra-cellularly labeled COINs and to trace the phospho-stat6 signaling pathway in U937 leukemia cells, demonstrating that COINs can be used to detect intracellular phosphorylation signaling events. These experiments demonstrate the importance of electron microscopy for analyzing the material-biology interface and for validating the attachment of nanoparticles on and in cells. Thus, electron microscope provides complementary imaging and spectroscopic information to current magnetic and SERS bio-detection technologies. (Abstract shortened by UMI.)

Prevalence of and Factors Associated with Negative Microscopic Diagnosis of Cutaneous Leishmaniasis in Rural Peru.

PubMed

Lamm, Ryan; Alves, Clark; Perrotta, Grace; Murphy, Meagan; Messina, Catherine; Sanchez, Juan F; Perez, Erika; Rosales, Luis Angel; Lescano, Andres G; Smith, Edward; Valdivia, Hugo; Fuhrer, Jack; Ballard, Sarah-Blythe

2018-06-04

Cutaneous leishmaniasis is endemic to South America where diagnosis is most commonly conducted via microscopy. Patients with suspected leishmaniasis were referred for enrollment by the Ministry of Health (MoH) in Lima, Iquitos, Puerto Maldonado, and several rural areas of Peru. A 43-question survey requesting age, gender, occupation, characterization of the lesion(s), history of leishmaniasis, and insect-deterrent behaviors was administered. Polymerase chain reaction (PCR) was conducted on lesion materials at the Naval Medical Research Unit No. 6 in Lima, and the results were compared with those obtained by the MoH using microscopy. Factors associated with negative microscopy and positive PCR results were identified using χ 2 test, t -test, and multivariate logistic regression analyses. Negative microscopy with positive PCR occurred in 31% (123/403) of the 403 cases. After adjusting for confounders, binary multivariate logistic regression analyses revealed that negative microscopy with positive PCR was associated with patients who were male (adjusted OR = 1.93 [1.06-3.53], P = 0.032), had previous leishmaniasis (adjusted OR = 2.93 [1.65-5.22], P < 0.0001), had larger lesions (adjusted OR = 1.02 [1.003-1.03], P = 0.016), and/or had a longer duration between lesion appearance and PCR testing (adjusted OR = 1.12 [1.02-1.22], P = 0.017). Future research should focus on further exploration of these underlying variables, discovery of other factors that may be associated with negative microscopy diagnosis, and the development and implementation of improved testing in endemic regions.

Continuum generation in ultra high numerical aperture fiber with application to multiphoton microscopy

NASA Astrophysics Data System (ADS)

Sayler, Nicholas

Nonlinear microscopy benefits from broadband laser sources, enabling efficient excitation of an array of fluorophores, for example. This work demonstrates broadening of a narrow band input pulse (6 nm to 40 nm) centered at 1040 nm with excellent shot-to-shot stability. In a preliminary demonstration, multiphoton imaging with pulses from the fiber is performed. In particular second harmonic imaging of corn starch is performed.

Microstructure and nutrient distribution in oats: influence on quality

NASA Astrophysics Data System (ADS)

Miller, S. Shea; Frégeau-Reid, Judith

2009-05-01

Oats have long been recognized as having superior quality among cereals with respect to protein and lipid composition as well as soluble dietary fibre (β-glucan). The microstructure and chemistry of oats influence oat quality, and thus are determinants of the end products derived from oats. Light and scanning electron microscopies have been used to elucidate microstructure and nutrient distribution in oats. The influence of variation in these parameters on oat quality can be demonstrated, from milling through to oat products for consumption. Milling quality is determined in part by hull architecture. SEM examination of oat hulls can help predict ease of dehulling, which affects the efficiency and economics of oat milling. In addition to protein and lipid, β-glucan is an important nutritional component of oats. Fluorescence microscopy can reveal both the relative amount and distribution of β-glucan in oat kernels. Consumption of oats or oat products containing β-glucan has been shown to have beneficial effects on carbohydrate and lipid metabolism. These health benefits have generated a demand for new and palatable ways to incorporate oats into the diet as consumer demand increases. To help meet this need, we have been investigating the use of micronized naked oats as a whole grain to be cooked and consumed as a rice alternative. Different varieties of naked oats had dramatically different acceptance levels from a sensory panel. SEM of the pericarp, light microscopy of the endosperm, and analyses of starch properties of the different varieties revealed differences that corresponded with sensory data.

In vivo multiphoton microscopy of deep tissue with gradient index lenses

NASA Astrophysics Data System (ADS)

Levene, Michael J.; Dombeck, Daniel A.; Williams, Rebecca M.; Skoch, Jesse; Hickey, Gregory A.; Kasischke, Karl A.; Molloy, Raymond P.; Ingelsson, Martin; Stern, Edward A.; Klucken, Jochen; Bacskai, Brian J.; Zipfel, Warren R.; Hyman, Bradley T.; Webb, Watt W.

2004-06-01

Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.

Recent Advances in Fiber Lasers for Nonlinear Microscopy

PubMed Central

Xu, C.; Wise, F. W.

2013-01-01

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

Ophthalmic imaging using multiphoton microscopy

NASA Astrophysics Data System (ADS)

Teng, Shu-Wen; Peng, Ju-Li; Lin, Huei-Hsing; Wu, Hai-Yin; Lo, Wen; Sun, Yen; Lin, Wei-Chou; Lin, Sung-Jan; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

2005-04-01

This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in ophthalmologic imaging. Without the introduction of extrinsic fluorescence molecules, multiphoton induced autofluorescence and second harmonic generation signals can be used to obtain useful structural information of normal and diseased corneas. Our work can potentially lead to the in vivo application of multiphoton microscopy in investigating corneal physiology and pathologies.

Methods for visualising active microbial benzene degraders in in situ microcosms.

PubMed

Schurig, Christian; Mueller, Carsten W; Höschen, Carmen; Prager, Andrea; Kothe, Erika; Beck, Henrike; Miltner, Anja; Kästner, Matthias

2015-01-01

Natural attenuation maybe a cost-efficient option for bioremediation of contaminated sites but requires knowledge about the activity of degrading microbes under in situ conditions. In order to link microbial activity to the spatial distribution of contaminant degraders, we combined the recently improved in situ microcosm approach, so-called 'direct-push bacterial trap' (DP-BACTRAP), with nano-scale secondary ion mass spectrometry (NanoSIMS) analysis on samples from contaminated constructed wetlands. This approach is based on initially sterile microcosms amended with (13)C-labelled benzene as a source of carbon and energy for microorganisms. The microcosms were introduced directly in the constructed wetland, where they were colonised by indigenous microorganisms from the sediment. After incubation in the field, the samples were analysed by NanoSIMS, scanning electron microscopy (SEM) and fluorescence microscopy in order to visualise (13)C-labelled microbial biomass on undisturbed samples from the microcosms. With the approach developed, we successfully visualised benzene-degrading microbes on solid materials with high surface area by means of NanoSIMS. Moreover, we could demonstrate the feasibility of NanoSIMS analysis of unembedded porous media with a highly complex topography, which was frequently reasoned to not lead to sufficient results.

Sputtered Pd as Hydrogen Storage for a Chip-Integrated Microenergy System

PubMed Central

Slavcheva, E.; Ganske, G.; Schnakenberg, U.

2014-01-01

The work presents a research on preparation and physical and electrochemical characterisation of dc magnetron sputtered Pd films envisaged for application as hydrogen storage in a chip-integrated hydrogen microenergy system. The influence of the changes in the sputtering pressure on the surface structure, morphology, and roughness was analysed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and atomic force microscopy (AMF). The electrochemical activity towards hydrogen adsorption/desorption and formation of PdH were investigated in 0.5 M H2SO4 using the methods of cyclic voltammetry and galvanostatic polarisation. The changes in the electrical properties of the films as a function of the sputtering pressure and the level of hydrogenation were evaluated before and immediately after the electrochemical charging tests, using a four-probe technique. The research resulted in establishment of optimal sputter regime, ensuring fully reproducible Pd layers with highly developed surface, moderate porosity, and mechanical stability. Selected samples were integrated as hydrogen storage in a newly developed unitized microenergy system and tested in charging (water electrolysis) and discharging (fuel cell) operative mode at ambient conditions demonstrating a stable recycling performance. PMID:24516356

Molecular responses of cells to 2-mercapto-1-methylimidazole gold nanoparticles (AuNPs)-mmi: investigations of histone methylation changes

NASA Astrophysics Data System (ADS)

Polverino, Arianna; Longo, Angela; Donizetti, Aldo; Drongitis, Denise; Frucci, Maria; Schiavo, Loredana; Carotenuto, Gianfranco; Nicolais, Luigi; Piscopo, Marina; Vitale, Emilia; Fucci, Laura

2014-07-01

While nanomedicine has an enormous potential to improve the precision of specific therapy, the ability to efficiently deliver these materials to regions of disease in vivo remains limited. In this study, we describe analyses of (AuNPs)-mmi cellular intake via fluorescence microscopy and its effects on H3K4 and H3K9 histone dimethylation. Specifically, we studied the level of H3K4 dimethylation in serving the role of an epigenetic marker of euchromatin, and of H3K9 dimethylation as a marker of transcriptional repression in four different cell lines. We analyzed histone di-methyl-H3K4 and di-methyl-H3K9 using either variable concentrations of nanoparticles or variable time points after cellular uptake. The observed methylation effects decreased consistently with decreasing (AuNPs)-mmi concentrations. Fluorescent microscopy and a binarization algorithm based on a thresholding process with RGB input images demonstrated the continued presence of (AuNPs)-mmi in cells at the lowest concentration used. Furthermore, our results show that the treated cell line used is able to rescue the untreated cell phenotype.

Biomediated Precipitation of Calcium Carbonate in a Slightly Acidic Hot Spring

NASA Astrophysics Data System (ADS)

Jiang, L.

2015-12-01

A slightly acidic hot spring named "Female Tower" (T=73.5 °C, pH=6.64) is located in the Jifei Geothermal Field, Yunnan Province, Southwest China. The precipitates in the hot spring are composed of large amounts of calcite, aragonite, and sulfur. Scanning electron microscopy (SEM) analyses revealed that the microbial mats were formed of various coccoid, rod-shaped, and filamentous microbes. Transmission electron microscopy (TEM) showed that the intracellular sulfur granules were commonly associated with these microbes. A culture-independent molecular phylogenetic analysis demonstrated that the majority of the bacteria in the spring were sulfur-oxidizing bacteria. In the spring water, H2S concentration was up to 60 ppm, while SO42- concentration was only about 10 ppm. We speculated that H2S might be utilized by sulfur-oxidizing bacteria in this hot spring water, leading to the intracellular formation of sulfur granules. In the meantime, this reaction increased the pH in the micron-scale microdomains, which fostered the precipitation of calcium carbonate in the microbial mats. The results of this study indicated that the sulfur-oxidizing bacteria could play an important role in calcium carbonate precipitation in slightly acidic hot spring environments.

HRTEMFringeAnalyzer a free python module for an automated analysis of fringe pattern in transmission electron micrographs.

PubMed

Alxneit, Ivo

2018-03-30

A python module (HRTEMFringeAnalyzer) is reported to evaluate the local crystallinity of samples from high-resolution transmission electron microscopy images in a mostly automated fashion. The user only selects the size of a square analyser window and a step size which translates the window in the micrograph. Together they define the resolution of the results obtained. Regions where fringe patterns are visible are identified and their lattice spacing d and direction ϕ as well as the corresponding mean errors σ determined. 1/σd is proportional to the coherence length of the structure, whereas σφ is a measure of how well the direction of the fringes is defined. Maps of these four indicators are computed. The performance of the program is demonstrated on two very different samples: ill-crystalline carbon deposits on a coked Ni/LFNO (reduced LaFe 0.8 Ni 0.2 O3±δ) catalyst and well-crystallized nanoparticles of zinc doped ceria. In the latter case, the automatic segmentation of large aggregates into individual crystalline domains is achieved by ϕ maps. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Imaging cervical cytology with scanning near-field optical microscopy (SNOM) coupled with an IR-FEL.

PubMed

Halliwell, Diane E; Morais, Camilo L M; Lima, Kássio M G; Trevisan, Julio; Siggel-King, Michele R F; Craig, Tim; Ingham, James; Martin, David S; Heys, Kelly A; Kyrgiou, Maria; Mitra, Anita; Paraskevaidis, Evangelos; Theophilou, Georgios; Martin-Hirsch, Pierre L; Cricenti, Antonio; Luce, Marco; Weightman, Peter; Martin, Francis L

2016-07-12

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (≥0.2 μm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 μm to 30 μm due to the optical diffraction limit.

Imaging cervical cytology with scanning near-field optical microscopy (SNOM) coupled with an IR-FEL

PubMed Central

Halliwell, Diane E.; Morais, Camilo L. M.; Lima, Kássio M. G.; Trevisan, Julio; Siggel-King, Michele R. F.; Craig, Tim; Ingham, James; Martin, David S.; Heys, Kelly A.; Kyrgiou, Maria; Mitra, Anita; Paraskevaidis, Evangelos; Theophilou, Georgios; Martin-Hirsch, Pierre L.; Cricenti, Antonio; Luce, Marco; Weightman, Peter; Martin, Francis L.

2016-01-01

Cervical cancer remains a major cause of morbidity and mortality among women, especially in the developing world. Increased synthesis of proteins, lipids and nucleic acids is a pre-condition for the rapid proliferation of cancer cells. We show that scanning near-field optical microscopy, in combination with an infrared free electron laser (SNOM-IR-FEL), is able to distinguish between normal and squamous low-grade and high-grade dyskaryosis, and between normal and mixed squamous/glandular pre-invasive and adenocarcinoma cervical lesions, at designated wavelengths associated with DNA, Amide I/II and lipids. These findings evidence the promise of the SNOM-IR-FEL technique in obtaining chemical information relevant to the detection of cervical cell abnormalities and cancer diagnosis at spatial resolutions below the diffraction limit (≥0.2 μm). We compare these results with analyses following attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy; although this latter approach has been demonstrated to detect underlying cervical atypia missed by conventional cytology, it is limited by a spatial resolution of ~3 μm to 30 μm due to the optical diffraction limit. PMID:27406404

The transfer of titanium dioxide nanoparticles from the host plant to butterfly larvae through a food chain

PubMed Central

Kubo-Irie, Miyoko; Yokoyama, Masaaki; Shinkai, Yusuke; Niki, Rikio; Takeda, Ken; Irie, Masaru

2016-01-01

This study aimed to examine the transfer of nanoparticles within a terrestrial food chain. Oviposited eggs of the swallowtail butterfly (Atrophaneura alcinous) were hatched on the leaves of the host plant (Aristolochia debilis), and the root stock and root hairs were submerged in a suspension of 10 μg/ml titanium dioxide nanoparticles (TiO2-NPs) in a 100 ml bottle. The presence of TiO2-NPs in the veins of the leaves was confirmed by X-ray analytical microscopy (X-ray AM). The hatched 1st instar larvae fed on the leaves to moult into 2nd instar larvae. Small agglomerates of TiO2-NPs less than 150 nm in diameter were identified in the vascular tissue of the exposed plant, the midgut and the excreta of the larvae by transmission electron microscopy. The image of Ti elemental mapping by X-ray AM was analysed with the quantitative spatial information mapping (QSIM) technique. The results demonstrated that TiO2-NPs were transferred from the plant to the larvae and they were disseminated throughout the environment via larval excreta. PMID:27030539

Strain Effects in Epitaxial VO2 Thin Films on Columnar Buffer-Layer TiO2/Al2O3 Virtual Substrates.

PubMed

Breckenfeld, Eric; Kim, Heungsoo; Burgess, Katherine; Charipar, Nicholas; Cheng, Shu-Fan; Stroud, Rhonda; Piqué, Alberto

2017-01-18

Epitaxial VO 2 /TiO 2 thin film heterostructures were grown on (100) (m-cut) Al 2 O 3 substrates via pulsed laser deposition. We have demonstrated the ability to reduce the semiconductor-metal transition (SMT) temperature of VO 2 to ∼44 °C while retaining a 4 order of magnitude SMT using the TiO 2 buffer layer. A combination of electrical transport and X-ray diffraction reciprocal space mapping studies help examine the specific strain states of VO 2 /TiO 2 /Al 2 O 3 heterostructures as a function of TiO 2 film growth temperatures. Atomic force microscopy and transmission electron microscopy analyses show that the columnar microstructure present in TiO 2 buffer films is responsible for the partially strained VO 2 film behavior and subsequently favorable transport characteristics with a lower SMT temperature. Such findings are of crucial importance for both the technological implementation of the VO 2 system, where reduction of its SMT temperature is widely sought, as well as the broader complex oxide community, where greater understanding of the evolution of microstructure, strain, and functional properties is a high priority.

Template-Free Hydrothermal Synthesis, Mechanism, and Photocatalytic Properties of Core-Shell CeO2 Nanospheres

NASA Astrophysics Data System (ADS)

Li, Huijie; Meng, Fanming; Gong, Jinfeng; Fan, Zhenghua; Qin, Rui

2018-03-01

CeO2 nanospheres with the core-shell nanostructure have been successfully synthesized by a template-free hydrothermal method. The structures, morphologies and optical properties of core-shell CeO2 nanospheres were analyzed by X-ray diffraction (XRD), TG, Fourier transform infrared spectroscopy, XRD, EDS, SAED, scanning electron microscopy and transmission electron microscopy, UV-Vis diffuse reflectance spectra, Raman analyses. The degradation efficiencies of core-shell CeO2 nanospheres for methyl orange were as high as 93.49, 95.67 and 98.28% within 160 min, and the rates of photo degradation of methyl orange by core-shell CeO2 nanospheres under UV-light were 0.01693, 0.01782 and 0.02375 min-1. Methyl orange was degraded in photocatalytic oxidation processes, which mainly gave the credit to a large number of reactive species including h+, surface superoxide species ·O2 -, and ·OH radicals. The core-shell structure, small crystallite size and the conversion between Ce3+ and Ce4+ of CeO2 nanospheres were of importance for its catalytic activity. These results demonstrated the possibility of improving the efficient catalysts of the earth abundant CeO2 catalysts.

Hollow Pd/MOF Nanosphere with Double Shells as Multifunctional Catalyst for Hydrogenation Reaction.

PubMed

Wan, Mingming; Zhang, Xinlu; Li, Meiyan; Chen, Bo; Yin, Jie; Jin, Haichao; Lin, Lin; Chen, Chao; Zhang, Ning

2017-10-01

A new type of hollow nanostructure featured double metal-organic frameworks shells with metal nanoparticles (MNPs) is designed and fabricated by the methods of ship in a bottle and bottle around the ship. The nanostructure material, hereinafter denoted as Void@HKUST-1/Pd@ZIF-8, is confirmed by the analyses of photograph, transmission electron microscopy, scanning electron microscopy, powder X-ray diffraction, inductively coupled plasma, and N 2 sorption. It possesses various multifunctionally structural characteristics such as hollow cavity which can improve mass transfer, the adjacent of the inner HKUST-1 shell to the void which enables the matrix of the shell to host and well disperse MNPs, and an outer ZIF-8 shell which acts as protective layer against the leaching of MNPs and a sieve to guarantee molecular-size selectivity. This makes the material eligible candidates for the heterogeneous catalyst. As a proof of concept, the liquid-phase hydrogenation of olefins with different molecular sizes as a model reaction is employed. It demonstrates the efficient catalytic activity and size-selectivity of Void@HKUST-1/Pd@ZIF-8. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amplitude quantification in contact-resonance-based voltage-modulated force spectroscopy

NASA Astrophysics Data System (ADS)

Bradler, Stephan; Schirmeisen, André; Roling, Bernhard

2017-08-01

Voltage-modulated force spectroscopy techniques, such as electrochemical strain microscopy and piezoresponse force microscopy, are powerful tools for characterizing electromechanical properties on the nanoscale. In order to correctly interpret the results, it is important to quantify the sample motion and to distinguish it from the electrostatic excitation of the cantilever resonance. Here, we use a detailed model to describe the cantilever dynamics in contact resonance measurements, and we compare the results with experimental values. We show how to estimate model parameters from experimental values and explain how they influence the sensitivity of the cantilever with respect to the excitation. We explain the origin of different crosstalk effects and how to identify them. We further show that different contributions to the measured signal can be distinguished by analyzing the correlation between the resonance frequency and the measured amplitude. We demonstrate this technique on two representative test samples: (i) ferroelectric periodically poled lithium niobate, and (ii) the Na+-ion conducting soda-lime float glass. We extend our analysis to higher cantilever bending modes and show that non-local electrostatic excitation is strongly reduced in higher bending modes due to the nodes in the lever shape. Based on our analyses, we present practical guidelines for quantitative imaging.

High performance Sb2S3/carbon composite with tailored artificial interface as an anode material for sodium ion batteries

NASA Astrophysics Data System (ADS)

Choi, Jeong-Hee; Ha, Chung-Wan; Choi, Hae-Young; Shin, Heon-Cheol; Lee, Sang-Min

2017-11-01

The electrochemical comparison between Sb2S3 and its composite with carbon (Sb2S3/C) involved by sodium ion carrier are explained by enhanced kinetics, particularly with respect to improved interfacial conductivity by surface modulation by carbon. Sb2S3 and Sb2S3/C are synthesized by a high energy mechanical milling process. The successful synthesis of these materials is confirmed with X-ray diffraction (XRD), scanning electron microscopy, and transmission electron microscopy (TEM). As an anode material for sodium ion batteries, Sb2S3 exhibits an initial sodiation/desodiation capacity of 1,021/523 mAh g-1 whereas the Sb2S3/C composite exhibits a higher reversible capacity (642 mAh g-1). Furthermore, the cycle performance and rate capability of the Sb2S3/C composite are estimated to be much better than those of Sb and Sb2S3. Electrochemical impedance spectroscopy analysis shows that the Sb2S3/C composite exhibited charge transfer resistance and surface film resistance much lower than Sb2S3. X-ray photoelectron spectroscopy analyses of both electrodes demonstrate that NaF layer on Sb2S3/C composite electrode leads to the better electrochemical performances. In order to clarify the electrochemical reaction mechanism, ex-situ XRD based on differential capacity plots and ex-situ HR-TEM analyses of the Sb2S3/C composite electrode are carried out and its reaction mechanism was established.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

PubMed Central

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

2016-01-01

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

PubMed

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

2016-10-04

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

In situ growth of ceramic quantum dots in polyaniline host via water vapor flow diffusion as potential electrode materials for energy applications

NASA Astrophysics Data System (ADS)

Mombrú, Dominique; Romero, Mariano; Faccio, Ricardo; Castiglioni, Jorge; Mombrú, Alvaro W.

2017-06-01

In situ preparation of polyaniline-ceramic nanocomposites has recently demonstrated that the electrical properties are highly improved with respect to the typical ex situ preparations. In this report, we present for the first time, to the best of our knowledge, the in situ growth of titanium oxide quantum dots in polyaniline host via water vapor flow diffusion as an easily adaptable route to prepare other ceramic-polymer nanocomposites. The main relevance of this method is the possibility to prepare ceramic quantum dots from alkoxide precursors using water vapor flow into any hydrophobic polymer host and to achieve good homogeneity and size-control. In addition, we perform full characterization by means of high-resolution transmission electron microscopy, X-ray powder diffraction, small angle X-ray scattering, thermogravimetric and calorimetric analyses, confocal Raman microscopy and impedance spectroscopy analyses. The presence of the polymer host and interparticle Coulomb repulsive interactions was evaluated as an influence for the formation of 3-8 nm equally-sized quantum dots independently of the concentration. The polyaniline polaron population showed an increase for the quantum dots diluted regime and the suppression at the concentrated regime, ascribed to the formation of chemical bonds at the interface, which was confirmed by theoretical simulations. In agreement with the previous observation, the in situ growth of ceramic quantum dots in polyaniline host via water vapor flow diffusion could be very useful as a novel approach to prepare electrode materials for energy conversion and storage applications.

MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

PubMed

Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

2016-03-08

The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Facile synthesis of lithium sulfide nanocrystals for use in advanced rechargeable batteries

DOE PAGES

Li, Xuemin; Wolden, Colin A.; Ban, Chunmei; ...

2015-12-03

This work reports a new method of synthesizing anhydrous lithium sulfide (L i2S) nanocrystals and demonstrates their potential as cathode materials for advanced rechargeable batteries. Li 2S is synthesized by reacting hydrogen sulfide (H 2S) with lithium naphthalenide (Li-NAP), a thermodynamically spontaneous reaction that proceeds to completion rapidly at ambient temperature and pressure. The process completely removes H 2S, a major industrial waste, while cogenerating 1,4-dihydronaphthalene, itself a value-added chemical that can be used as liquid fuel. The phase purity, morphology, and homogeneity of the resulting nanopowders were confirmed by X-ray diffraction and scanning electron microscopy. The synthesized Li 2Smore » nanoparticles (100 nm) were assembled into cathodes, and their performance was compared to that of cathodes fabricated using commercial Li 2S micropowders (1–5 μm). As a result, electrochemical analyses demonstrated that the synthesized Li 2S were superior in terms of (dis)charge capacity, cycling stability, output voltage, and voltage efficiency.« less

The low-temperature method for study of coniferous tissues in the environmental scanning electron microscope.

PubMed

Neděla, Vilém; Tihlaříková, Eva; Hřib, Jiří

2015-01-01

The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella. © 2014 Wiley Periodicals, Inc.

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

PubMed

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

Structural and optical investigation on the wings of Idea malabarica (Moore, 1877).

PubMed

Sackey, Juliet; Nuru, Zebib Y; Sone, Bertrand Tumbain; Maaza, Malik

2017-02-01

The nanostructures on the wings of Idea malabarica (Moore, 1877) were analysed using scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy, Fourier transform-infrared spectroscopy, and reflectance measurements. The chemical and morphological analyses revealed the chitin-based intricate nanostructures. The influence of the nanostructures on the wetting characteristics of the wing was investigated using optical imaging. Applying the Maxwell-Garnet approximation to the porosities within the nanostructures, the refractive indices, which relate the reflectance response, were estimated. It was concluded that the colour seen on the wings of the Idea malabarica originate from the nanostructural configurations of the chitin-based structures and the embedded pigment.

Acousto-optical tunable filter for combined wideband, spectral, and optical coherence microscopy.

PubMed

Machikhin, Alexander S; Pozhar, Vitold E; Viskovatykh, Alexander V; Burmak, Ludmila I

2015-09-01

A multimodal technique for inspection of microscopic objects by means of wideband optical microscopy, spectral microscopy, and optical coherence microscopy is described, implemented, and tested. The key feature is the spectral selection of light in the output arm of an interferometer with use of the specialized imaging acousto-optical tunable filter. In this filter, two interfering optical beams are diffracted via the same ultrasound wave without destruction of interference image structure. The basic requirements for the acousto-optical tunable filter are defined, and mathematical formulas for calculation of its parameters are derived. Theoretical estimation of the achievable accuracy of the 3D image reconstruction is presented and experimental proofs are given. It is demonstrated that spectral imaging can also be accompanied by measurement of the quantitative reflectance spectra. Examples of inspection of optically transparent and nontransparent samples demonstrate the applicability of the technique.

The OptIPuter microscopy demonstrator: enabling science through a transatlantic lightpath

PubMed Central

Ellisman, M.; Hutton, T.; Kirkland, A.; Lin, A.; Lin, C.; Molina, T.; Peltier, S.; Singh, R.; Tang, K.; Trefethen, A.E.; Wallom, D.C.H.; Xiong, X.

2009-01-01

The OptIPuter microscopy demonstrator project has been designed to enable concurrent and remote usage of world-class electron microscopes located in Oxford and San Diego. The project has constructed a network consisting of microscopes and computational and data resources that are all connected by a dedicated network infrastructure using the UK Lightpath and US Starlight systems. Key science drivers include examples from both materials and biological science. The resulting system is now a permanent link between the Oxford and San Diego microscopy centres. This will form the basis of further projects between the sites and expansion of the types of systems that can be remotely controlled, including optical, as well as electron, microscopy. Other improvements will include the updating of the Microsoft cluster software to the high performance computing (HPC) server 2008, which includes the HPC basic profile implementation that will enable the development of interoperable clients. PMID:19487201

The OptIPuter microscopy demonstrator: enabling science through a transatlantic lightpath.

PubMed

Ellisman, M; Hutton, T; Kirkland, A; Lin, A; Lin, C; Molina, T; Peltier, S; Singh, R; Tang, K; Trefethen, A E; Wallom, D C H; Xiong, X

2009-07-13

The OptIPuter microscopy demonstrator project has been designed to enable concurrent and remote usage of world-class electron microscopes located in Oxford and San Diego. The project has constructed a network consisting of microscopes and computational and data resources that are all connected by a dedicated network infrastructure using the UK Lightpath and US Starlight systems. Key science drivers include examples from both materials and biological science. The resulting system is now a permanent link between the Oxford and San Diego microscopy centres. This will form the basis of further projects between the sites and expansion of the types of systems that can be remotely controlled, including optical, as well as electron, microscopy. Other improvements will include the updating of the Microsoft cluster software to the high performance computing (HPC) server 2008, which includes the HPC basic profile implementation that will enable the development of interoperable clients.

Production of intensive negative lithium beam with caesium sputter-type ion source

NASA Astrophysics Data System (ADS)

Lobanov, Nikolai R.

2018-01-01

Compounds of lithium oxide, hydroxide and carbonate, mixed with silver, were prepared for use as a cathode in caesium-sputter ion source. The intention was to determine the procedure which would produce the highest intensity negative lithium beams over extended period and with maximum stability. The chemical composition and properties of the samples were analysed using mass-spectrometry, optical microscopy, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Analyses (EDX) and Raman spectroscopy. These analyses showed that the chemical transformations with components resulted from pressing, storage and bake out were qualitatively in agreement with expectations. Intensive negative lithium ion beams >1 μA were delivered using cathodes fabricated from materials with multicomponent chemical composition when the following conditions were met: (i) use of components with moderate enthalpy of formation; (ii) low moisture content at final stage of cathode production and (iii) small concentration of water molecules in hydrate phase in the cathode mixture.

Evaluation of a new automated microscopy urine sediment analyser - sediMAX conTRUST®.

PubMed

Bogaert, Laura; Peeters, Bart; Billen, Jaak

2017-04-01

This study evaluated the performance of the stand-alone sediMAX conTRUST (77Elektronika, Budapest, Hungary) analyser as an alternative to microscopic analysis of urine. The validation included a precision, carry-over, categorical correlation and diagnostic performance study with manual phase-contrast microscopy as reference method. A total of 260 routine urine samples were assessed. The within-run precision was much better at higher concentrations than at very low concentrations. The precision met our predefined limits for all the elements at the different concentrations, with the exception of the lowest RBC, the WBC, pathological casts and crystals count. There was no sample carry-over. The analyser showed good categorical agreement with manual microscopy for RBC and WBC counts, moderate agreement for yeast cells, crystals and squamous epithelial cells and bad agreement for non-squamous epithelial cells, bacteria and casts. Diagnostic performance was satisfying only for RBC, WBC and yeast cells. The number of false negative results was acceptable (≤4%) for all elements after connecting the sediMAX conTRUST with an automatic strip reader (AutionMAX) and after implementation of review rules. We conclude that the sediMAX conTRUST should be used as a screening tool in combination with an automatic strip reader, for the identification of normal samples. Therefore, adequate review rules should be defined. Manual microscopy is still required in 'flagged' pathological samples. Despite the poor analytical performance on pathological samples, the images on the screen can be used for interpretation without the microscope and can be stored as PDF-documents for archiving the results.

Analysis of pigments in polychromes by use of laser induced breakdown spectroscopy and Raman microscopy

NASA Astrophysics Data System (ADS)

Castillejo, M.; Martín, M.; Silva, D.; Stratoudaki, T.; Anglos, D.; Burgio, L.; Clark, R. J. H.

2000-09-01

Two laser-based analytical techniques, Laser Induced Breakdown Spectroscopy (LIBS) and Raman microscopy, have been used for the identification of pigments on a polychrome from the Rococo period. Detailed spectral data are presented from analyses performed on a fragment of a gilded altarpiece from the church of Escatrón, Zaragoza, Spain. LIBS measurements yielded elemental analytical data which suggest the presence of certain pigments and, in addition, provide information on the stratigraphy of the paint layers. Identification of most pigments and of the materials used in the preparation layer was performed by Raman microscopy.

Multiple microscopic approaches demonstrate linkage between chromoplast architecture and carotenoid composition in diverse Capsicum annuum fruit.

PubMed

Kilcrease, James; Collins, Aaron M; Richins, Richard D; Timlin, Jerilyn A; O'Connell, Mary A

2013-12-01

Increased accumulation of specific carotenoids in plastids through plant breeding or genetic engineering requires an understanding of the limitations that storage sites for these compounds may impose on that accumulation. Here, using Capsicum annuum L. fruit, we demonstrate directly the unique sub-organellar accumulation sites of specific carotenoids using live cell hyperspectral confocal Raman microscopy. Further, we show that chromoplasts from specific cultivars vary in shape and size, and these structural variations are associated with carotenoid compositional differences. Live-cell imaging utilizing laser scanning confocal (LSCM) and confocal Raman microscopy, as well as fixed tissue imaging by scanning and transmission electron microscopy (SEM and TEM), all demonstrated morphological differences with high concordance for the measurements across the multiple imaging modalities. These results reveal additional opportunities for genetic controls on fruit color and carotenoid-based phenotypes. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

PubMed

Baroux, Célia; Schubert, Veit

2018-01-01

In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

In-situ observation of recrystallization in an AlMgScZr alloy using confocal laser scanning microscopy

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

Taendl, J., E-mail: johannes.taendl@tugraz.atl; Nambu, S.; Orthacker, A.

2015-10-15

In this work we present a novel in-situ approach to study the recrystallization behavior of age hardening alloys. We use confocal laser scanning microscopy (CLSM) at 400 °C to investigate the static recrystallization of an AlMg4Sc0.4Zr0.12 alloy in-situ. The results are combined with electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) analyses. It was found that CLSM is a powerful tool to visualize both the local initiation and temporal sequence of recrystallization. After fast nucleation and initial growth, the grain growth rate decreases and the grain boundary migration stops after some minutes due to Zener pinning from Al{sub 3}(Sc,Zr)more » precipitates produced during the heat treatment. EBSD and TEM analyses confirm both the boundary movements and the particle-boundary interactions. - Highlights: • First time that CLSM is used to study recrystallization in-situ. • The start and end of recrystallization can be directly observed. • The procedure is easy to apply and requires only simple data interpretation. • In-situ observations on the surface correlate to modifications inside the bulk. • In-situ observations correlate to EBSD and EFTEM analyses.« less

Comparison of nitrogen adsorption and transmission electron microscopy analyses for structural characterization of carbon nanotubes

NASA Astrophysics Data System (ADS)

Abbaslou, Reza Malek; Vosoughi, Vahid; Dalai, Ajay K.

2017-10-01

Carbon nanotubes (CNTs) are different from other porous substrates such as activated carbon due to their high external surfaces. This structural feature can lead in some uncertainties in the results of nitrogen adsorption analysis for characterization of CNTs. In this paper, the results of microscopic analyses and nitrogen adsorption method for characterization of carbon nanotubes were compared. Five different types of CNTs with different structures were either synthesized or purchased. The CNT samples were characterized by high resolution transmission electron microscopy (HRTEM), scanning electron microscopy (SEM) and N2 adsorption analysis. The comparisons between the results from the microscopic analyses and N2 adsorption showed that the total pore volume and BET surface measurements include the internal and external porosity of CNTs. Therefore, the interpretation of N2 adsorption data required accurate TEM analysis. In addition, the evaluation of pore size distribution curves from all CNT samples in this study and several instances in the literature revealed the presence of a common peak in the range of 2-5 nm. This peak does not explain the inner pore size distribution. The presence of this common peak can be attributed to the strong adsorption of N2 on the junction of touched and crossed nanotubes.

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.

Gold Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

NASA Technical Reports Server (NTRS)

Vikram, Chandra S.; Witherow, William K.

2000-01-01

We report what is believed to be the first experimental demonstration of gold coating by a chemical baking process on tapered fiber tips used in near-field scanning optical microscopy. Many tips can be simultaneously coated.

Chemical and biological characterization of emissions from coal- and oil-fired power plants.

PubMed Central

Ahlberg, M; Berghem, L; Nordberg, G; Persson, S A; Rudling, L; Steen, B

1983-01-01

Emission samples were obtained from two medium-sized power plants, one fired with oil and the other with pulverized coal. Particles obtained by a miniscale plume stack gas sampler (MIPSGAS), simulating the dilution process in the plume, were subjected to detailed physical, chemical and biological characterization. Studies by scanning electron microscopy and by Coulter counter demonstrated that the particles from the oil-fired boiler were considerably larger than the particles from the coal-fired boiler. Chemical analyses revealed more organic substances and more S, Ni, V, in the oil than in the coal particles. The latter contained a larger proportion of Al, Si, Cl, K, Ca, Ti, Mn, Fe, Se, Rb, Y, Zr, Ba and Pb. Biological testing revealed a greater acute and subacute toxicity by the intratracheal route in the hamster, a greater toxicity to alveolar macrophages and a greater lung retention of BaP coated on the particles from oil combustion than on those from coal combustion. In another sampling line, employed simultaneously with the MIPSGAS-particulate sampler, the total emissions were collected, i.e., both particle and gas phase. These samples were used for chemical analyses and Ames mutagenicity test. Analyses of specific PAHs in emissions from both plants demonstrated that concentrations were below the detection limit (less than 4 ng/m3 of benzo(a)pyrene), which is in accord with an efficient combustion of the fuel. The mutagenicity of the samples were below the detection limit of the mutagenicity assay. Images FIGURE 4. FIGURE 5. PMID:6825622

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

Internal quality assurance in diagnostic microbiology: A simple approach for insightful data

PubMed Central

Scherz, Valentin; Durussel, Christian

2017-01-01

Given the importance of microbiology results on patient care, high quality standards are expected. Internal quality assurance (IQA) could mitigate the limitations of internal quality control, competency assessment and external quality assurance, adding a longitudinal insight, including pre- and post-analytical steps. Here, we implemented an IQA program in our clinical microbiology facilities with blind resubmission of routine samples during 22 months. One-hundred-and-twenty-one out of 123 (98.4%) serological analyses and 112 out of 122 (91.8%) molecular analyses were concordant. Among the discordances in molecular biology analyses, 6 results were low positive samples that turned out negative, likely due to stochastic repartition of nucleic acids. Moreover, one identified retranscription error led us to implement automated results transmission from the Applied Biosystems instruments to the laboratory information system (LIS). Regarding Gram stain microscopy, 560 out of 745 (75.2%) of compared parameters were concordant. As many as 67 out of 84 (79.8%) pairs of culture results were similar, including 16 sterile pairs, 27 having identical identification or description and semi-quantification and 24 only showing variations in semi-quantification with identical description or identification of colonies. Seventeen pairs had diverging identification or description of colonies. Culture was twice only done for one member of the pairs. Regarding antibiotic susceptibility testing, a major discrepancy was observed in 5 out of 48 results (10.4%). In conclusion, serological tests were highly reproducible. Molecular diagnosis also revealed to be robust except when the amounts of nucleic acids present in the sample were close to the limits of detection. Conventional microbiology was less robust with major discrepancies reaching 39.5% of the samples for microscopy. Similarly, culture and antibiotic susceptibility testing were prone to discrepancies. This work was ground for reconsidering multiples aspects of our practices and demonstrates the importance of IQA to complete the other quality management procedures. PMID:29135992

Internal quality assurance in diagnostic microbiology: A simple approach for insightful data.

PubMed

Scherz, Valentin; Durussel, Christian; Greub, Gilbert

2017-01-01

Given the importance of microbiology results on patient care, high quality standards are expected. Internal quality assurance (IQA) could mitigate the limitations of internal quality control, competency assessment and external quality assurance, adding a longitudinal insight, including pre- and post-analytical steps. Here, we implemented an IQA program in our clinical microbiology facilities with blind resubmission of routine samples during 22 months. One-hundred-and-twenty-one out of 123 (98.4%) serological analyses and 112 out of 122 (91.8%) molecular analyses were concordant. Among the discordances in molecular biology analyses, 6 results were low positive samples that turned out negative, likely due to stochastic repartition of nucleic acids. Moreover, one identified retranscription error led us to implement automated results transmission from the Applied Biosystems instruments to the laboratory information system (LIS). Regarding Gram stain microscopy, 560 out of 745 (75.2%) of compared parameters were concordant. As many as 67 out of 84 (79.8%) pairs of culture results were similar, including 16 sterile pairs, 27 having identical identification or description and semi-quantification and 24 only showing variations in semi-quantification with identical description or identification of colonies. Seventeen pairs had diverging identification or description of colonies. Culture was twice only done for one member of the pairs. Regarding antibiotic susceptibility testing, a major discrepancy was observed in 5 out of 48 results (10.4%). In conclusion, serological tests were highly reproducible. Molecular diagnosis also revealed to be robust except when the amounts of nucleic acids present in the sample were close to the limits of detection. Conventional microbiology was less robust with major discrepancies reaching 39.5% of the samples for microscopy. Similarly, culture and antibiotic susceptibility testing were prone to discrepancies. This work was ground for reconsidering multiples aspects of our practices and demonstrates the importance of IQA to complete the other quality management procedures.

LASER BIOLOGY: Peculiarities of studying an isolated neuron by the method of laser interference microscopy

NASA Astrophysics Data System (ADS)

Yusipovich, Alexander I.; Novikov, Sergey M.; Kazakova, Tatiana A.; Erokhova, Liudmila A.; Brazhe, Nadezda A.; Lazarev, Grigory L.; Maksimov, Georgy V.

2006-09-01

Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.

Magnetoelectric force microscopy based on magnetic force microscopy with modulated electric field.

PubMed

Geng, Yanan; Wu, Weida

2014-05-01

We present the realization of a mesoscopic imaging technique, namely, the Magnetoelectric Force Microscopy (MeFM), for visualization of local magnetoelectric effect. The basic principle of MeFM is the lock-in detection of local magnetoelectric response, i.e., the electric field-induced magnetization, using magnetic force microscopy. We demonstrate MeFM capability by visualizing magnetoelectric domains on single crystals of multiferroic hexagonal manganites. Results of several control experiments exclude artifacts or extrinsic origins of the MeFM signal. The parameters are tuned to optimize the signal to noise ratio.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Multispectral confocal microscopy images and artificial neural nets to monitor the photosensitizer uptake and degradation in Candida albicans cells

NASA Astrophysics Data System (ADS)

Romano, Renan A.; Pratavieira, Sebastião.; da Silva, Ana P.; Kurachi, Cristina; Guimarães, Francisco E. G.

2017-07-01

This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

The role of biomineralization in microbiologically influenced corrosion

NASA Technical Reports Server (NTRS)

Little, B.; Wagner, P.; Hart, K.; Ray, R.; Lavoie, D.; Nealson, K.; Aguilar, C.

1998-01-01

Synthetic iron oxides (goethite, alpha-FeO.OH; hematite, Fe2O3; and ferrihydrite, Fe(OH)3) were used as model compounds to simulate the mineralogy of surface films on carbon steel. Dissolution of these oxides exposed to pure cultures of the metal-reducing bacterium, Shewanella putrefaciens, was followed by direct atomic absorption spectroscopy measurement of ferrous iron coupled with microscopic analyses using confocal laser scanning and environmental scanning electron microscopies. During an 8-day exposure the organism colonized mineral surfaces and reduced solid ferric oxides to soluble ferrous ions. Elemental composition, as monitored by energy dispersive x-ray spectroscopy, indicated mineral replacement reactions with both ferrihydrite and goethite as iron reduction occurred. When carbon steel electrodes were exposed to S. putrefaciens, microbiologically influenced corrosion was demonstrated electrochemically and microscopically.

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

PubMed

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

2012-11-01

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

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

PubMed Central

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

2012-01-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

Comparison of quartz crystallographic preferred orientations identified with optical fabric analysis, electron backscatter and neutron diffraction techniques.

PubMed

Hunter, N J R; Wilson, C J L; Luzin, V

2017-02-01

Three techniques are used to measure crystallographic preferred orientations (CPO) in a naturally deformed quartz mylonite: transmitted light cross-polarized microscopy using an automated fabric analyser, electron backscatter diffraction (EBSD) and neutron diffraction. Pole figure densities attributable to crystal-plastic deformation are variably recognizable across the techniques, particularly between fabric analyser and diffraction instruments. Although fabric analyser techniques offer rapid acquisition with minimal sample preparation, difficulties may exist when gathering orientation data parallel with the incident beam. Overall, we have found that EBSD and fabric analyser techniques are best suited for studying CPO distributions at the grain scale, where individual orientations can be linked to their source grain or nearest neighbours. Neutron diffraction serves as the best qualitative and quantitative means of estimating the bulk CPO, due to its three-dimensional data acquisition, greater sample area coverage, and larger sample size. However, a number of sampling methods can be applied to FA and EBSD data to make similar approximations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Comprehensive study of unexpected microscope condensers formed in sample arrangements commonly used in optical microscopy.

PubMed

Desai, Darshan B; Aldawsari, Mabkhoot Mudith S; Alharbi, Bandar Mohammed H; Sen, Sanchari; Grave de Peralta, Luis

2015-09-01

We show that various setups for optical microscopy which are commonly used in biomedical laboratories behave like efficient microscope condensers that are responsible for observed subwavelength resolution. We present a series of experiments and simulations that reveal how inclined illumination from such unexpected condensers occurs when the sample is perpendicularly illuminated by a microscope's built-in white-light source. In addition, we demonstrate an inexpensive add-on optical module that serves as an efficient and lightweight microscope condenser. Using such add-on optical module in combination with a low-numerical-aperture objective lens and Fourier plane imaging microscopy technique, we demonstrate detection of photonic crystals with a period nearly eight times smaller than the Rayleigh resolution limit.

In Situ Synthesis of Reduced Graphene Oxide and Gold Nanocomposites for Nanoelectronics and Biosensing.

PubMed

Dong, Xiaochen; Huang, Wei; Chen, Peng

2011-12-01

In this study, an in situ chemical synthesis approach has been developed to prepare graphene-Au nanocomposites from chemically reduced graphene oxide (rGO) in aqueous media. UV-Vis absorption, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy were used to demonstrate the successful attachment of Au nanoparticles to graphene sheets. Configured as field-effect transistors (FETs), the as-synthesized single-layered rGO-Au nanocomposites exhibit higher hole mobility and conductance when compared to the rGO sheets, promising its applications in nanoelectronics. Furthermore, we demonstrate that the rGO-Au FETs are able to label-freely detect DNA hybridization with high sensitivity, indicating its potentials in nanoelectronic biosensing.

In Situ Synthesis of Reduced Graphene Oxide and Gold Nanocomposites for Nanoelectronics and Biosensing

PubMed Central

2011-01-01

In this study, an in situ chemical synthesis approach has been developed to prepare graphene–Au nanocomposites from chemically reduced graphene oxide (rGO) in aqueous media. UV–Vis absorption, atomic force microscopy, scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy were used to demonstrate the successful attachment of Au nanoparticles to graphene sheets. Configured as field-effect transistors (FETs), the as-synthesized single-layered rGO-Au nanocomposites exhibit higher hole mobility and conductance when compared to the rGO sheets, promising its applications in nanoelectronics. Furthermore, we demonstrate that the rGO-Au FETs are able to label-freely detect DNA hybridization with high sensitivity, indicating its potentials in nanoelectronic biosensing. PMID:27502682

TiO2-Based Photocatalytic Geopolymers for Nitric Oxide Degradation

PubMed Central

Strini, Alberto; Roviello, Giuseppina; Ricciotti, Laura; Ferone, Claudio; Messina, Francesco; Schiavi, Luca; Corsaro, Davide; Cioffi, Raffaele

2016-01-01

This study presents an experimental overview for the development of photocatalytic materials based on geopolymer binders as catalyst support matrices. Particularly, geopolymer matrices obtained from different solid precursors (fly ash and metakaolin), composite systems (siloxane-hybrid, foamed hybrid), and curing temperatures (room temperature and 60 °C) were investigated for the same photocatalyst content (i.e., 3% TiO2 by weight of paste). The geopolymer matrices were previously designed for different applications, ranging from insulating (foam) to structural materials. The photocatalytic activity was evaluated as NO degradation in air, and the results were compared with an ordinary Portland cement reference. The studied matrices demonstrated highly variable photocatalytic performance depending on both matrix constituents and the curing temperature, with promising activity revealed by the geopolymers based on fly ash and metakaolin. Furthermore, microstructural features and titania dispersion in the matrices were assessed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analyses. Particularly, EDS analyses of sample sections indicated segregation effects of titania in the surface layer, with consequent enhancement or depletion of the catalyst concentration in the active sample region, suggesting non-negligible transport phenomena during the curing process. The described results demonstrated that geopolymer binders can be interesting catalyst support matrices for the development of photocatalytic materials and indicated a large potential for the exploitation of their peculiar features. PMID:28773634

Mass-specific scattering coefficient for natural minerogenic particle populations: particle size distribution effect and closure analyses.

PubMed

Peng, Feng; Effler, Steve W

2012-05-01

The relationship between the particulate scattering coefficient (b(p)) and the concentration of suspended particulate matter (SPM), as represented by the mass-specific scattering coefficient of particulates (b(p)*=b(p)/SPM), depends on particle size distribution (PSD). This dependence is quantified for minerogenic particle populations in this paper through calculations of b(p)* for common minerals as idealized populations (monodispersed spheres); contemporaneous measurements of b(p), SPM, and light-scattering attributes of mineral particles with scanning electron microscopy interfaced with automated image and x-ray analyses (SAX), for a connected stream-reservoir system where minerogenic particles dominate b(p); and estimates of b(p) and its size dependency (through SAX results-driven Mie theory calculations), particle volume concentration, and b(p)*. Modest changes in minerogenic PSDs are shown to result in substantial variations in b(p)*. Good closure of the SAX-based estimates of b(p) and particle volume concentration with bulk measurements is demonstrated. Converging relationships between b(p)* and particle size, developed from three approaches, were well described by power law expressions.

Chromatographic fingerprinting as a strategy to identify regulated plants in illegal herbal supplements.

PubMed

Custers, D; Van Praag, N; Courselle, P; Apers, S; Deconinck, E

2017-03-01

Erectile dysfunction (ED) is a sexual disorder characterized by the inability to achieve or maintain a sufficiently rigid erection. Despite the availability of non-invasive oral treatment options, many patients turn to herbal alternatives. Furthermore, herbal supplements are increasingly gaining popularity in industrialized countries and, as a consequence, quality control is a highly important issue. Unfortunately, this is not a simple task since plants are often crushed and mixed with other plants, which complicates their identification by usage of classical approaches such as microscopy. The aim of this study was to explore the potential use of chromatographic fingerprinting to identify plants present in herbal preparations intended for the treatment of ED. To achieve this goal, a HPLC-PDA and a HPLC-MS method were developed, using a full factorial experimental design in order to acquire characteristic fingerprints of three plants which are potentially beneficial for treating ED: Epimedium spp., Pausinystalia yohimbe and Tribulus terrestris. The full factorial design demonstrated that for all three plant references a C8 column (250mm×4.6mm; 5µm particle size) is best suited; methanol and an ammonium formate buffer (pH 3) were found to be the best constituents for the mobile phase. The suitability of this strategy was demonstrated by analysing several self-made triturations in three different botanical matrices, which mimic the influential effects that could be expected when analysing herbal supplements. To conclude, this study demonstrates that chromatographic fingerprinting could provide a useful means to identify plants in a complex herbal mixture. Copyright © 2016 Elsevier B.V. All rights reserved.

Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1

PubMed Central

Sironen, Anu; Kotaja, Noora; Mulhern, Howard; Wyatt, Todd A.; Sisson, Joseph H.; Pavlik, Jacqueline A.; Miiluniemi, Mari; Fleming, Mark D.; Lee, Lance

2011-01-01

Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice. PMID:21715716

Peculiarities of studying an isolated neuron by the method of laser interference microscopy

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

Yusipovich, Alexander I; Kazakova, Tatiana A; Erokhova, Liudmila A

2006-09-30

Actual aspects of using a new method of laser interference microscopy (LIM) for studying nerve cells are discussed. The peculiarities of the LIM display of neurons are demonstrated by the example of isolated neurons of a pond snail Lymnaea stagnalis. A comparative analysis of the images of the cell and subcellular structures of a neuron obtained by the methods of interference microscopy, optical transmission microscopy, and confocal microscopy is performed. Various aspects of the application of LIM for studying the lateral dimensions and internal structure of the cytoplasm and organelles of a neuron in cytology and cell physiology are discussed.more » (laser biology)« less

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Synthesis of calcium carbonate in alkali solution based on graphene oxide and reduced graphene oxide

NASA Astrophysics Data System (ADS)

Yaseen, Sarah Abduljabbar; Yiseen, Ghadah Abdaljabar; Li, Zongjin

2018-06-01

This paper reports a new approach of producing CaCO3 particles in alkali solution. CaCO3 particles with pure calcite structure were obtained from the reaction of water-dispersed graphene oxide (GO) or reduced graphene oxide (rGO) with either Ca(OH)2 or CaO. In Fourier Transform Infrared (FTIR) spectra, the pure calcite structure was demonstrated by fundamental bands at 1425 (ν3), 873 (ν2), and 712 cm-1 (ν4). The Raman spectra showed the characteristic peak of calcite structure at 1085 cm-1 (ν1). X-ray diffraction pattern (XRD) and X-ray photoelectron spectroscopy (XPS) analyses further confirmed that only the pure calcite phase of CaCO3 was formed in both synthesis approaches. Scanning electron microscopy (SEM), Energy dispersive X-ray analyzer (EDX), and High-resolution transmission electron microscopy (HRTEM) also confirmed that distorted cubic and rhombic calcite particles were obtained with GO, while the pine flower-like and flower-like particles were obtained with rGO, and the average crystallite sizes varied from 26 to 44 nm. The mechanism of the reaction was investigated and it was found that the decomposition of oxygen functional groups on the surface of GO or rGO in certain alkaline media to release CO, CO2, and water was a key process as the released CO2 further reacted with OH- and Ca2+ to form CaCO3. This demonstrated that both GO and rGO could be used as main reactants for the synthesis of calcite.

Anaerobic choline metabolism in microcompartments promotes growth and swarming of P roteus mirabilis

PubMed Central

Jameson, Eleanor; Fu, Tiantian; Brown, Ian R.; Paszkiewicz, Konrad; Purdy, Kevin J.

2015-01-01

Summary Gammaproteobacteria are important gut microbes but only persist at low levels in the healthy gut. The ecology of G ammaproteobacteria in the gut environment is poorly understood. Here, we demonstrate that choline is an important growth substrate for representatives of G ammaproteobacteria. Using P roteus mirabilis as a model, we investigate the role of choline metabolism and demonstrate that the cut C gene, encoding a choline‐trimethylamine lyase, is essential for choline degradation to trimethylamine by targeted mutagenesis of cut C and subsequent complementation experiments. P roteus mirabilis can rapidly utilize choline to enhance growth rate and cell yield in broth culture. Importantly, choline also enhances swarming‐associated colony expansion of P . mirabilis under anaerobic conditions on a solid surface. Comparative transcriptomics demonstrated that choline not only induces choline‐trimethylamine lyase but also genes encoding shell proteins for the formation of bacterial microcompartments. Subsequent analyses by transmission electron microscopy confirmed the presence of such novel microcompartments in cells cultivated in liquid broth and hyper‐flagellated swarmer cells from solid medium. Together, our study reveals choline metabolism as an adaptation strategy for P . mirabilis and contributes to better understand the ecology of this bacterium in health and disease. PMID:26404097

Role of phi cells and the endodermis under salt stress in Brassica oleracea.

PubMed

Fernandez-Garcia, N; Lopez-Perez, L; Hernandez, M; Olmos, E

2009-01-01

Phi cell layers were discovered in the 19th century in a small number of species, including members of the Brassicaceae family. A mechanical role was first suggested for this structure; however, this has never been demonstrated. The main objective of the present work was to analyse the ultrastructure of phi cells, their influence on ion movement from the cortex to the stele, and their contribution to salt stress tolerance in Brassica oleracea. Transmission electron microscopy and X-ray microanalysis studies were used to analyse the subcellular structure and distribution of ions in phi cells and the endodermis under salt stress. Ion movement was analysed using lanthanum as an apoplastic tracer. The ultrastructural results confirm that phi cells are specialized cells showing cell wall ingrowths in the inner tangential cell walls. X-ray microanalysis confirmed a build-up of sodium. Phi thickenings were lignified and lanthanum moved periplasmically at this level. To the best of our knowledge, this is the first study reporting the possible role of the phi cells as a barrier controlling the movement of ions from the cortex to the stele. Therefore, the phi cell layer and endodermis seem to be regulating ion transport in Brassica oleracea under salt stress.

DESIGN NOTE: A modified Nanosurf scanning tunnelling microscope for ballistic electron emission microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

Appelbaum, Ian; Thompson, Pete; van Schendel, P. J. A.

2006-04-01

We describe the design and implementation of modifications to an ambient STM with a slip stick approach mechanism to create a system capable of ballistic electron emission microscopy (BEEM) and spectroscopy (BEES). These modifications require building a custom sample holder which operates as a high gain transimpedance preamplifier. Results of microscopy and spectroscopy using a Au/n-GaAs Schottky device demonstrate the effectiveness of our design.

Dark Field Microscopy for Analytical Laboratory Courses

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

Augspurger, Ashley E; Stender, Anthony S; Marchuk, Kyle

2014-06-10

An innovative and inexpensive optical microscopy experiment for a quantitative analysis or an instrumental analysis chemistry course is described. The students have hands-on experience with a dark field microscope and investigate the wavelength dependence of localized surface plasmon resonance in gold and silver nanoparticles. Students also observe and measure individual crystal growth during a replacement reaction between copper and silver nitrate. The experiment allows for quantitative, qualitative, and image data analyses for undergraduate students.

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.

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Automated analysis of high-content microscopy data with deep learning.

PubMed

Kraus, Oren Z; Grys, Ben T; Ba, Jimmy; Chong, Yolanda; Frey, Brendan J; Boone, Charles; Andrews, Brenda J

2017-04-18

Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Helium ion microscopy of Lepidoptera scales.

PubMed

Boden, Stuart A; Asadollahbaik, Asa; Rutt, Harvey N; Bagnall, Darren M

2012-01-01

In this report, helium ion microscopy (HIM) is used to study the micro and nanostructures responsible for structural color in the wings of two species of Lepidotera from the Papilionidae family: Papilio ulysses (Blue Mountain Butterfly) and Parides sesostris (Emerald-patched Cattleheart). Electronic charging of uncoated scales from the wings of these butterflies, due to the incident ion beam, is successfully neutralized, leading to images displaying a large depth-of-field and a high level of surface detail, which would normally be obscured by traditional coating methods used for scanning electron microscopy (SEM). The images are compared with those from variable pressure SEM, demonstrating the superiority of HIM at high magnifications. In addition, the large depth-of-field capabilities of HIM are exploited through the creation of stereo pairs that allows the exploration of the third dimension. Furthermore, the extraction of quantitative height information which matches well with cross-sectional transmission electron microscopy measurements from the literature is demonstrated. © Wiley Periodicals, Inc.

Spectral Demultiplexing in Holographic and Fluorescent On-chip Microscopy

NASA Astrophysics Data System (ADS)

Sencan, Ikbal; Coskun, Ahmet F.; Sikora, Uzair; Ozcan, Aydogan

2014-01-01

Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip.

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PubMed

Parsa, Kami; Schaudinn, Christoph; Gorur, Amita; Sedghizadeh, Parish P; Johnson, Thomas; Tse, David T; Costerton, John W

2010-01-01

To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

In vivo detection of hemoglobin oxygen saturation and carboxyhemoglobin saturation with multiwavelength photoacoustic microscopy.

PubMed

Chen, Zhongjiang; Yang, Sihua; Xing, Da

2012-08-15

A method for noninvasively detecting hemoglobin oxygen saturation (SO2) and carboxyhemoglobin saturation (SCO) in subcutaneous microvasculature with multiwavelength photoacoustic microscopy is presented. Blood samples mixed with different concentrations of carboxyhemoglobin were used to test the feasibility and accuracy of photoacoustic microscopy compared with the blood-gas analyzer. Moreover, fixed-point detection of SO2 and SCO in mouse ear was obtained, and the changes from normoxia to carbon monoxide hypoxia were dynamically monitored in vivo. Experimental results demonstrate that multiwavelength photoacoustic microscopy can detect SO2 and SCO, which has future potential clinical applications.

Electron microscopy study of gold nanoparticles deposited on transition metal oxides.

PubMed

Akita, Tomoki; Kohyama, Masanori; Haruta, Masatake

2013-08-20

Many researchers have investigated the catalytic performance of gold nanoparticles (GNPs) supported on metal oxides for various catalytic reactions of industrial importance. These studies have consistently shown that the catalytic activity and selectivity depend on the size of GNPs, the kind of metal oxide supports, and the gold/metal oxide interface structure. Although researchers have proposed several structural models for the catalytically active sites and have identified the specific electronic structures of GNPs induced by the quantum effect, recent experimental and theoretical studies indicate that the perimeter around GNPs in contact with the metal oxide supports acts as an active site in many reactions. Thus, it is of immense importance to investigate the detailed structures of the perimeters and the contact interfaces of gold/metal oxide systems by using electron microscopy at an atomic scale. This Account describes our investigation, at the atomic scale using electron microscopy, of GNPs deposited on metal oxides. In particular, high-resolution transmission electron microscopy (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) are valuable tools to observe local atomic structures, as has been successfully demonstrated for various nanoparticles, surfaces, and material interfaces. TEM can be applied to real powder catalysts as received without making special specimens, in contrast to what is typically necessary to observe bulk materials. For precise structure analyses at an atomic scale, model catalysts prepared by using well-defined single-crystalline substrates are also adopted for TEM observations. Moreover, aberration-corrected TEM, which has high spatial resolution under 0.1 nm, is a promising tool to observe the interface structure between GNPs and metal oxide supports including oxygen atoms at the interfaces. The oxygen atoms in particular play an important role in the behavior of gold/metal oxide interfaces, because they may participate in catalytic reaction steps. Detailed information about the interfacial structures between GNPs and metal oxides provides valuable structure models for theoretical calculations which can elucidate the local electronic structure effective for activating a reactant molecule. Based on our observations with HRTEM and HAADF-STEM, we report the detailed structure of gold/metal oxide interfaces.

TEM Study of Intergranular Fluid Distributions in Rocks at a Nanometer Scale

NASA Astrophysics Data System (ADS)

Hiraga, T.; Anderson, I. M.; Kohlstedt, D. L.

2002-12-01

The distribution of intergranular fluids in rocks plays an essential role in fluid migration and rock rheology. Structural and chemical analyses with sub-nanometer resolution is possible with transmission and scanning-transmission electron microscopy; therefore, it is possible to perform the fine-scale structural analyses required to determine the presence or absence of very thin fluid films along grain boundaries. For aqueous fluids in crustal rocks, Hiraga et al. (2001) observed a fluid morphology controlled by the relative values of the solid-solid and solid-fluid interfacial energies, which resulted in well-defined dihedral angles. Their high-resolution transmission electron microscopy (TEM) observations demonstrate that grain boundaries are tight even at a nanometer scale, consistent with the absence of aqueous fluid films. For partially molten ultra-mafic rocks, two conflicting conclusions have been reached: nanometer-thick melt films wet grain boundaries (Drury and Fitz Gerald 1996; De Kloe et al. 2000) versus essentially all grain boundaries are melt-free (Vaughan et al. 1982; Kohlstedt 1990). To resolve this conflict, Hiraga et al. (2002) examined grain boundaries in quenched partially molten peridotites. Their observations demonstrate the following: (i) Although a small fraction of the grains are separated by relatively thick (~1 μm) layers of melt, lattice fringe images obtained with a high-resolution TEM reveal that most of the remaining boundaries do not contain a thin amorphous phase. (ii) In addition, the composition of olivine-olivine grain boundaries was analyzed with a nano-beam analytical scanning TEM with a probe size of <2 nm. Although the grain boundaries contained no melt film, the concentration of Ca, Al and Ti were enhanced near the boundaries. The segregation of these elements to the grain boundaries formed enriched regions <7 nm wide. A similar pattern of chemical segregation was detected in subsolidus systems. Creep experiments on the partially molten rocks that were analyzed in this study reveal little weakening even at melt contents approaching 4 vol%, consistent with our observations of melt-free grain boundaries.

Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

PubMed

Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F

2014-03-01

Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires <1 min and <5 min for 2D and 3D images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

DOE PAGES

Ophus, Colin; Ciston, Jim; Pierce, Jordan; ...

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, makingmore » it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Ultimately, simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.« less

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry.

PubMed

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R; Chess, Jordan; McMorran, Benjamin J; Czarnik, Cory; Rose, Harald H; Ercius, Peter

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy

NASA Astrophysics Data System (ADS)

Bon, Pierre; Bourg, Nicolas; Lécart, Sandrine; Monneret, Serge; Fort, Emmanuel; Wenger, Jérôme; Lévêque-Fort, Sandrine

2015-07-01

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

PubMed Central

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

2016-01-01

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals. PMID:26923483

Carrier Density Modulation in Ge Heterostructure by Ferroelectric Switching

DOE PAGES

Ponath, Patrick; Fredrickson, Kurt; Posadas, Agham B.; ...

2015-01-14

The development of nonvolatile logic through direct coupling of spontaneous ferroelectric polarization with semiconductor charge carriers is nontrivial, with many issues, including epitaxial ferroelectric growth, demonstration of ferroelectric switching, and measurable semiconductor modulation. Here we report a true ferroelectric field effect carrier density modulation in an underlying Ge(001) substrate by switching of the ferroelectric polarization in the epitaxial c-axis-oriented BaTiO3 (BTO) grown by molecular beam epitaxy (MBE) on Ge. Using density functional theory, we demonstrate that switching of BTO polarization results in a large electric potential change in Ge. Aberration-corrected electron microscopy confirms the interface sharpness, and BTO tetragonality. Electron-energy-lossmore » spectroscopy (EELS) indicates the absence of any low permittivity interlayer at the interface with Ge. Using piezoelectric force microscopy (PFM), we confirm the presence of fully switchable, stable ferroelectric polarization in BTO that appears to be single domain. Using microwave impedance microscopy (MIM), we clearly demonstrate a ferroelectric field effect.« less

Simple and robust image-based autofocusing for digital microscopy.

PubMed

Yazdanfar, Siavash; Kenny, Kevin B; Tasimi, Krenar; Corwin, Alex D; Dixon, Elizabeth L; Filkins, Robert J

2008-06-09

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

Intravital Microscopy Imaging Approaches for Image-Guided Drug Delivery Systems

PubMed Central

Kirui, Dickson K.; Ferrari, Mauro

2016-01-01

Rapid technical advances in the field of non-linear microscopy have made intravital microscopy a vital pre-clinical tool for research and development of imaging-guided drug delivery systems. The ability to dynamically monitor the fate of macromolecules in live animals provides invaluable information regarding properties of drug carriers (size, charge, and surface coating), physiological, and pathological processes that exist between point-of-injection and the projected of site of delivery, all of which influence delivery and effectiveness of drug delivery systems. In this Review, we highlight how integrating intravital microscopy imaging with experimental designs (in vitro analyses and mathematical modeling) can provide unique information critical in the design of novel disease-relevant drug delivery platforms with improved diagnostic and therapeutic indexes. The Review will provide the reader an overview of the various applications for which intravital microscopy has been used to monitor the delivery of diagnostic and therapeutic agents and discuss some of their potential clinical applications. PMID:25901526

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically-important Chrysomya rufifacies (Diptera: Calliphoridae)*

PubMed Central

Flores, Danielle; Miller, Amy L.; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M.N.; Sung, Carl; Stokes, Alexander J.; Tomberlin, Jeffrey K.; Carter, David O.; Turner, Helen

2016-01-01

Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. PMID:27706817

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2

NASA Astrophysics Data System (ADS)

McConnell, Gail; Riis, Erling

2004-10-01

We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM.

Combining kriging, multispectral and multimodal microscopy to resolve malaria-infected erythrocyte contents.

PubMed

Dabo-Niang, S; Zoueu, J T

2012-09-01

In this communication, we demonstrate how kriging, combine with multispectral and multimodal microscopy can enhance the resolution of malaria-infected images and provide more details on their composition, for analysis and diagnosis. The results of this interpolation applied to the two principal components of multispectral and multimodal images illustrate that the examination of the content of Plasmodium falciparum infected human erythrocyte is improved. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

Simultaneous X-ray fluorescence and scanning X-ray diffraction microscopy at the Australian Synchrotron XFM beamline

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

Jones, Michael W. M.; Phillips, Nicholas W.; van Riessen, Grant A.

2016-08-11

Owing to its extreme sensitivity, quantitative mapping of elemental distributionsviaX-ray fluorescence microscopy (XFM) has become a key microanalytical technique. The recent realisation of scanning X-ray diffraction microscopy (SXDM) meanwhile provides an avenue for quantitative super-resolved ultra-structural visualization. The similarity of their experimental geometries indicates excellent prospects for simultaneous acquisition. Here, in both step- and fly-scanning modes, robust, simultaneous XFM-SXDM is demonstrated.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

PubMed

Lopes, Camila Madeira Tavares; Menna-Barreto, Rubem Figueiredo Sadok; Pavan, Márcio Galvão; Pereira, Mirian Cláudia De Souza; Roque, André Luiz R

2018-01-01

Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy

PubMed Central

Cisek, Richard; Wein, Marc N.; Turcotte, Raphaël; Haase, Christa; Yeh, Shu-Chi A.; Bharadwaj, Srinidhi; Raphael, Anthony P.; Paudel, Hari; Alt, Clemens; Liu, Tzu-Ming; Kronenberg, Henry M.; Lin, Charles P.

2017-01-01

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning. PMID:29065178

Fast and reliable method of conductive carbon nanotube-probe fabrication for scanning probe microscopy

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

Dremov, Vyacheslav, E-mail: dremov@issp.ac.ru; Fedorov, Pavel; Grebenko, Artem

2015-05-15

We demonstrate the procedure of scanning probe microscopy (SPM) conductive probe fabrication with a single multi-walled carbon nanotube (MWNT) on a silicon cantilever pyramid. The nanotube bundle reliably attached to the metal-covered pyramid is formed using dielectrophoresis technique from the MWNT suspension. It is shown that the dimpled aluminum sample can be used both for shortening/modification of the nanotube bundle by applying pulse voltage between the probe and the sample and for controlling the probe shape via atomic force microscopy imaging the sample. Carbon nanotube attached to cantilever covered with noble metal is suitable for SPM imaging in such modulationmore » regimes as capacitance contrast microscopy, Kelvin probe microscopy, and scanning gate microscopy. The majority of such probes are conductive with conductivity not degrading within hours of SPM imaging.« less

Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

NASA Astrophysics Data System (ADS)

Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

2015-07-01

Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

Eco-friendly synthesis of Solanum trilobatum extract-capped silver nanoparticles is compatible with good antimicrobial activities

NASA Astrophysics Data System (ADS)

Ramanathan, Santheraleka; Gopinath, Subash C. B.; Anbu, Periasamy; Lakshmipriya, Thangavel; Kasim, Farizul Hafiz; Lee, Choul-Gyun

2018-05-01

This study focused on the evaluation of antimicrobial activity of silver nanoparticles (AgNPs) after their green synthesis by means of a Solanum trilobatum bark extract. The obtained product with an intense surface plasmon resonance band at ∼442 nm with UV-visible spectroscopic analysis indicated the formation of AgNPs. The morphology of AgNPs was observed under transmission electron microscopy and field emission scanning electron microscopy, displayed that the eco-friendly synthesized AgNPs have a spherical shape with an average size of ∼25 nm in diameter. X-ray powder diffraction and selected area electron diffraction analyses confirmed that the AgNPs are crystalline in nature. Fourier transform infrared spectroscopy indicated that the AgNPs capped with active ingredients of the bark extract. X-ray photoelectron spectroscopy revealed elemental composition of the AgNPs. The performance of S. trilobatum bark extract-capped AgNPs in terms of inhibition of microbial growth was studied by disc diffusion and well diffusion assays. Eco-friendly synthesized S. trilobatum extract-capped AgNPs were found to possess enhanced antimicrobial properties: growth inhibition of gram-negative and gram-positive bacteria and of fungal species. These results demonstrated the potential applications of the indigenous medicinal plants to the field of nanotechnology.

Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer.

PubMed

Nooshadokht, Maryam; Kalantari-Khandani, Behjat; Sharifi, Iraj; Kamyabi, Hossein; Liyanage, Namal P M; Lagenaur, Laurel A; Kagnoff, Martin F; Singer, Steven M; Babaei, Zahra; Solaymani-Mohammadi, Shahram

2017-10-01

Human infection with the protozoan parasite Giardia duodenalis is one the most common parasitic diseases worldwide. Higher incidence rates of giardiasis have been reported from human subjects with multiple debilitating chronic conditions, including hypogammaglobulinemia and common variable immunodeficiency (CVID). In the current study, stool specimens were collected from 199 individuals diagnosed with HIV or cancer and immunocompetent subjects. The sensitivity of microscopy-based detection on fresh stool preparations, trichrome staining and stool antigen immunodetection for the diagnosis of G. duodenalis were 36%, 45.5% and 100%, respectively when compared with a highly sensitive stool-based PCR method as the gold standard. Further multilocus molecular analyses using glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) loci demonstrated that the AI genotype of G. duodenalis was the most prevalent, followed by the AII genotype and mixed (AI+B) infections. We concluded that stool antigen immunodetection-based immunoassays and stool-based PCR amplification had comparable sensitivity and specificity for the diagnosis of G. duodenalis infections in these populations. Stool antigen detection-based diagnostic modalities are rapid and accurate and may offer alternatives to conventional microscopy and PCR-based diagnostic methods for the diagnosis of G. duodenalis in human subjects living with HIV or cancer. Copyright © 2017. Published by Elsevier B.V.

Mucoadhesive films containing chitosan-coated nanoparticles: a new strategy for buccal curcumin release.

PubMed

Mazzarino, Letícia; Borsali, Redouane; Lemos-Senna, Elenara

2014-11-01

Mucoadhesive films containing curcumin-loaded nanoparticles were developed, aiming to prolong the residence time of the dosage form in the oral cavity and to increase drug absorption through the buccal mucosa. Films were prepared by the casting method after incorporation of curcumin-loaded chitosan-coated polycaprolactone nanoparticles into plasticized chitosan solutions. Different molar masses of mucoadhesive polysaccharide chitosan and concentrations of plasticizer glycerol were used to optimize the preparation conditions. Films obtained using medium and high molar mass chitosan were found to be homogeneous and flexible. Curcumin-loaded nanoparticles were uniformly distributed on the film surface, as evidenced by atomic force microscopy and high-resolution field-emission gun scanning electron microscopy (FEG-SEM) images. Analyses of film cross sections using FEG-SEM demonstrate the presence of nanoparticles inside the films. In addition, films proved to have a good rate of hydration in simulated saliva solution, displaying a maximum swelling of around 80% and in vitro prolonged-controlled delivery of curcumin. These results indicate that the mucoadhesive films containing nanoparticles offer a promising approach for buccal delivery of curcumin, which may be particularly useful in the treatment of periodontal diseases that require a sustained drug delivery. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Chemical-state-selective mapping at nanometer scale using synchrotron radiation and photoelectron emission microscopy.

PubMed

Hirao, Norie; Baba, Yuji; Sekiguchi, Tetsuhiro; Shimoyama, Iwao; Honda, Mitsunori

2010-01-01

For surface analyses of semiconductor devices and various functional materials, it has become indispensable to analyze valence states at nanometer scale due to the rapid developments of nanotechnology. Since a method for microscopic mapping dependent on the chemical bond states has not been established so far, we have developed a photoelectron emission microscopy (PEEM) system combined with synchrotron soft X-ray excitation. The samples investigated were Si/SiO(x) micro-patterns prepared by O(2)(+) ion implantation in Si(001) wafer using a mask. PEEM images excited by various photon energies around the Si K-edge were observed. The lateral spatial resolution of the system was about 41 nm. The brightness of each spot in PEEM images changed depending on the photon energy, due to the X-ray absorption intensity of the respective chemical state. Since the surface of this sample was topographically flat, it has been demonstrated that the present method can be applied to observations of the microscopic pattern, depending not on the morphology, but only on the valence states of silicon. We have also in-situ measured the changes of the PEEM images upon annealing, and elucidated the mechanism of the lateral diffusion of oxygen and valence states of silicon at the nanometer scale.

Micro- and macroscopic study on the porosity of marble as a function of temperature and impregnation

NASA Astrophysics Data System (ADS)

Malaga-Starzec, K.; Akesson, U.; Lindqvist, J. E.; Schouenborg, B.

2003-04-01

The thermal weathering of marble is demonstrated by the progressive granular decohesion that leads to an increased porosity and subsequently to loss of strength. In order to determine how temperature cycling initiates changes in the porosity of fresh and impregnated stones: two chemically and petrographically very different marble types were tested for water absorption and ultrasonic velocity propagation and analysed by fluorescence microscopy and nitrogen adsorption. The influence of the impregnation materials: GypStop P17 and P22, both silica sols with different particle size, on changes of the porosity was also evaluated. A separate long-term study of thermal expansion was additionally performed on fresh unimpregnated samples. The results indicated that inter-granular decohesion was more pronounced for the calictic marble than the dolomitic marble. The impregnation materials had a mitigating effect on the granular decohesion. Use of fluorescence microscopy, among the other methods, appears to give inexpensive and reliable information about internal structure of the marbles. A better understanding of the effect that temperature has on the porosity of marble could be used as a guide for election of suitable stone material for exterior use as well as an indication for appropriate conditioning of the samples before physical properties testing.

RAMAN spectroscopy imaging improves the diagnosis of papillary thyroid carcinoma

NASA Astrophysics Data System (ADS)

Rau, Julietta V.; Graziani, Valerio; Fosca, Marco; Taffon, Chiara; Rocchia, Massimiliano; Crucitti, Pierfilippo; Pozzilli, Paolo; Onetti Muda, Andrea; Caricato, Marco; Crescenzi, Anna

2016-10-01

Recent investigations strongly suggest that Raman spectroscopy (RS) can be used as a clinical tool in cancer diagnosis to improve diagnostic accuracy. In this study, we evaluated the efficiency of Raman imaging microscopy to discriminate between healthy and neoplastic thyroid tissue, by analyzing main variants of Papillary Thyroid Carcinoma (PTC), the most common type of thyroid cancer. We performed Raman imaging of large tissue areas (from 100 × 100 μm2 up to 1 × 1 mm2), collecting 38 maps containing about 9000 Raman spectra. Multivariate statistical methods, including Linear Discriminant Analysis (LDA), were applied to translate Raman spectra differences between healthy and PTC tissues into diagnostically useful information for a reliable tissue classification. Our study is the first demonstration of specific biochemical features of the PTC profile, characterized by significant presence of carotenoids with respect to the healthy tissue. Moreover, this is the first evidence of Raman spectra differentiation between classical and follicular variant of PTC, discriminated by LDA with high efficiency. The combined histological and Raman microscopy analyses allow clear-cut integration of morphological and biochemical observations, with dramatic improvement of efficiency and reliability in the differential diagnosis of neoplastic thyroid nodules, paving the way to integrative findings for tumorigenesis and novel therapeutic strategies.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds.

PubMed

Cao, Dechang; Xu, Huimin; Zhao, Yuanyuan; Deng, Xin; Liu, Yongxiu; Soppe, Wim J J; Lin, Jinxing

2016-12-01

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds. © 2016 American Society of Plant Biologists. All Rights Reserved.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds1

PubMed Central

Xu, Huimin; Liu, Yongxiu; Soppe, Wim J.J.; Lin, Jinxing

2016-01-01

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds. PMID:27760880

Careful stoichiometry monitoring and doping control during the tunneling interface growth of an n + InAs(Si)/p + GaSb(Si) Esaki diode

NASA Astrophysics Data System (ADS)

El Kazzi, S.; Alian, A.; Hsu, B.; Verhulst, A. S.; Walke, A.; Favia, P.; Douhard, B.; Lu, W.; del Alamo, J. A.; Collaert, N.; Merckling, C.

2018-02-01

In this work, we report on the growth of pseudomorphic and highly doped InAs(Si)/GaSb(Si) heterostructures on p-type (0 0 1)-oriented GaSb substrate and the fabrication and characterization of n+/p+ Esaki tunneling diodes. We particularly study the influence of the Molecular Beam Epitaxy shutter sequences on the structural and electrical characteristics of InAs(Si)/GaSb(Si) Esaki diodes structures. We use real time Reflection High Electron Diffraction analysis to monitor different interface stoichiometry at the tunneling interface. With Atomic Force Microscopy, X-ray diffraction and Transmission Electron Microscopy analyses, we demonstrate that an "InSb-like" interface leads to a sharp and defect-free interface exhibiting high quality InAs(Si) crystal growth contrary to the "GaAs-like" one. We then prove by means of Secondary Ion Mass Spectroscopy profiles that Si-diffusion at the interface allows the growth of highly Si-doped InAs/GaSb diodes without any III-V material deterioration. Finally, simulations are conducted to explain our electrical results where a high Band to Band Tunneling (BTBT) peak current density of Jp = 8 mA/μm2 is achieved.

Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

PubMed Central

Krieger, Viktoria; Liebl, David; Zhang, Yuying; Rajashekar, Roopa; Chlanda, Petr; Giesker, Katrin; Chikkaballi, Deepak; Hensel, Michael

2014-01-01

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella. PMID:25254663

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

PubMed

Rubino, L; Di Franco, A; Russo, M

2000-01-01

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Three-dimensional modeling and quantitative analysis of gap junction distributions in cardiac tissue.

PubMed

Lackey, Daniel P; Carruth, Eric D; Lasher, Richard A; Boenisch, Jan; Sachse, Frank B; Hitchcock, Robert W

2011-11-01

Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

Extended vapor-liquid-solid growth of silicon carbide nanowires.

PubMed

Rajesh, John Anthuvan; Pandurangan, Arumugam

2014-04-01

We developed an alloy catalytic method to explain extended vapor-liquid-solid (VLS) growth of silicon carbide nanowires (SiC NWs) by a simple thermal evaporation of silicon and activated carbon mixture using lanthanum nickel (LaNi5) alloy as catalyst in a chemical vapor deposition process. The LaNi5 alloy binary phase diagram and the phase relationships in the La-Ni-Si ternary system were play a key role to determine the growth parameters in this VLS mechanism. Different reaction temperatures (1300, 1350 and 1400 degrees C) were applied to prove the established growth process by experimentally. Scanning electron microscopy and transmission electron microscopy studies show that the crystalline quality of the SiC NWs increases with the temperature at which they have been synthesized. La-Ni alloyed catalyst particles observed on the top of the SiC NWs confirms that the growth process follows this extended VLS mechanism. The X-ray diffraction and confocal Raman spectroscopy analyses demonstrate that the crystalline structure of the SiC NWs was zinc blende 3C-SiC. Optical property of the SiC NWs was investigated by photoluminescence technique at room temperature. Such a new alloy catalytic method may be extended to synthesis other one-dimensional nanostructures.

Curcumin Induces Pancreatic Adenocarcinoma Cell Death via Reduction of the Inhibitors of Apoptosis

PubMed Central

Osterman, Carlos J. Díaz; Gonda, Amber; Stiff, TessaRae; Sigaran, Ulysses; Valenzuela, Malyn May Asuncion; Bennit, Heather R. Ferguson; Moyron, Ron B.; Khan, Salma; Wall, Nathan R.

2015-01-01

Objectives The inhibitor of apoptosis (IAP) proteins are critical modulators of chemotherapeutic resistance in various cancers. To address the alarming emergence of chemotherapeutic resistance in pancreatic cancer, we investigated the efficacy of the turmeric derivative curcumin in reducing IAP protein and mRNA expression resulting in pancreatic cancer cell death. Methods The pancreatic adenocarcinoma cell line PANC-1 was used to assess curcumin’s effects in pancreatic cancer. Curcumin uptake was measured by spectral analysis and fluorescence microscopy. AlamarBlue and Trypan blue exclusion assays were used to determine PANC-1 cell viability following curcumin treatment. Visualization of PANC-1 cell death was performed using Hoffman Modulation Contrast microscopy. Western blot and PCR analyses were used to evaluate curcumin’s effects on IAP protein and mRNA expression. Results Curcumin enters PANC-1 cells and is ubiquitously present within the cell following treatment. Furthermore, curcumin reduces cell viability and induces morphological changes characteristic of cell death. Additionally, curcumin decreases IAP protein and mRNA expression in PANC-1 cells. Conclusions These data demonstrate that PANC-1 cells are sensitive to curcumin treatment. Furthermore, curcumin as a potential therapeutic tool for overcoming chemotherapeutic resistance mediated by IAPs, supports a role for curcumin as part of the therapeutic approach for pancreatic cancer. PMID:26348467

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.

Novel highly porous magnetic hydrogel beads composed of chitosan and sodium citrate: an effective adsorbent for the removal of heavy metals from aqueous solutions.

PubMed

Pu, Shengyan; Ma, Hui; Zinchenko, Anatoly; Chu, Wei

2017-07-01

This research focuses on the removal of heavy metal ions from aqueous solutions using magnetic chitosan hydrogel beads as a potential sorbent. Highly porous magnetic chitosan hydrogel (PMCH) beads were prepared by a combination of in situ co-precipitation and sodium citrate cross-linking. Fourier transform infrared spectroscopy indicated that the high sorption efficiency of metal cations is attributable to the hydroxyl, amino, and carboxyl groups in PMCH beads. Thermogravimetric analysis demonstrated that introducing Fe 3 O 4 nanoparticles increases the thermal stability of the adsorbent. Laser confocal microscopy revealed highly uniform porous structure of the resultant PMCH beads, which contained a high moisture content (93%). Transmission electron microscopy micrographs showed that the Fe 3 O 4 nanoparticles, with a mean diameter of 5 ± 2 nm, were well dispersed inside the chitosan beads. Batch adsorption experiments and adsorption kinetic analysis revealed that the adsorption process obeys a pseudo-second-order model. Isotherm data were satisfactorily described by the Langmuir equation, and the maximum adsorption capacity of the adsorbent was 84.02 mg/g. Energy-dispersive X-ray spectroscopy and X-ray photoelectron spectra analyses were performed to confirm the adsorption of Pb 2+ and to identify the adsorption mechanism.

Development and characterization of monoclonal antibody to the lymphocystis disease virus of Japanese flounder Paralichthys olivaceus isolated from China.

PubMed

Cheng, Shunfeng; Zhan, Wenbin; Xing, Jing; Sheng, Xiuzhen

2006-08-01

Lymphocystis disease virus (LCDV) can infect, both naturally and experimentally, about 100 different teleost fish species. In this study, LCDV was purified using differential and gradient centrifugation from skin tumours of Japanese flounder Paralichthys olivaceus. A panel of five monoclonal antibodies (Mabs) against LCDV were produced by immunization of Balb/c mice with purified virus preparations. Analysed by the indirect enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), Western blot and immunogold electron microscopy (IEM), they showed specificity for LCDV. Immunofluorescent studies showed that the specific fluorescence signals appeared at the peripheral zone of hypertrophied cells cytoplasm where was the cytoplasmic inclusion bodies location and many of them formed ribbon-shaped. Western blot analysis demonstrated that two Mabs 1D7 and 2B6 reacted specifically to a single protein with an approximately molecular weight of 116kDa, Mab 3G3 reacted with two LCDV proteins at molecular mass of approximately 116 and 90kDa. Immunogold transmission electron-microscopy provided visualized evidence that the epitopes recognized by these Mabs were located on the outer surface of virions. The Mabs characterized should prove useful for developing LCDV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Photosynthetic microorganism-mediated synthesis of akaganeite (beta-FeOOH) nanorods.

PubMed

Brayner, Roberta; Yéprémian, Claude; Djediat, Chakib; Coradin, Thibaud; Herbst, Fréderic; Livage, Jacques; Fiévet, Fernand; Couté, Alain

2009-09-01

Common Anabaena and Calothrix cyanobacteria and Klebsormidium green algae are shown to form intracellularly akaganeite beta-FeOOH nanorods of well-controlled size and unusual morphology at room temperature. X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy X-ray energy dispersive spectrometry (SEM-EDS) analyses are used to investigate particle structure, size, and morphology. A mechanism involving iron-siderophore complex formation is proposed and compared with iron biomineralization in magnetotactic bacteria.

Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway.

PubMed

Markov, A G; Falchuk, E L; Kruglova, N M; Radloff, J; Amasheh, S

2016-01-01

Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Subsurface microscopy of interconnect layers of an integrated circuit.

PubMed

Köklü, F Hakan; Unlü, M Selim

2010-01-15

We apply the NA-increasing lens technique to confocal and wide-field backside microscopy of integrated circuits. We demonstrate 325 nm (lambda(0)/4) lateral spatial resolution while imaging metal structures located inside the interconnect layer of an integrated circuit. Vectorial field calculations are presented justifying our findings.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

Influence of a fluoridated medium with different pHs on commercially pure titanium-based implants.

PubMed

Sartori, Rafael; Correa, Cassia Bellotto; Marcantonio, Elcio; Vaz, Luis Geraldo

2009-02-01

The objective of this study was to assess the influence of a fluoride medium with different pHs on the corrosion resistance of three commercially pure titanium-based dental implant commercial brands, under scanning electron microscopy (SEM) and EDS. Forty-two dental implants, from three commercial brands, were used. Five years of regular use of mouth rinsing, with NaF 1500 ppm content and two different pHs, were simulated by immersing the specimens into that medium for 184 hours. SEM and EDS analyses demonstrated no evidence of corrosion on the specimens' surfaces after being submitted to fluoride ions or incorporation of fluoride ions to the set surface. It was possible to conclude that both the fluoride concentration and the pH of the solutions did not exert any influence upon implant corrosion resistance.

Classification and recognition of texture collagen obtaining by multiphoton microscope with neural network analysis

NASA Astrophysics Data System (ADS)

Wu, Shulian; Peng, Yuanyuan; Hu, Liangjun; Zhang, Xiaoman; Li, Hui

2016-01-01

Second harmonic generation microscopy (SHGM) was used to monitor the process of chronological aging skin in vivo. The collagen structures of mice model with different ages were obtained using SHGM. Then, texture feature with contrast, correlation and entropy were extracted and analysed using the grey level co-occurrence matrix. At last, the neural network tool of Matlab was applied to train the texture of collagen in different statues during the aging process. And the simulation of mice collagen texture was carried out. The results indicated that the classification accuracy reach 85%. Results demonstrated that the proposed approach effectively detected the target object in the collagen texture image during the chronological aging process and the analysis tool based on neural network applied the skin of classification and feature extraction method is feasible.

Chirality controlled responsive self-assembled nanotubes in water† †Electronic supplementary information (ESI) available: Detailed experimental procedures and analyses, UV-vis absorption and CD spectroscopy, cryo-TEM and widefield microscopy. See DOI: 10.1039/c6sc02935c Click here for additional data file.

PubMed Central

van Dijken, D. J.; Štacko, P.; Stuart, M. C. A.; Browne, W. R.

2017-01-01

The concept of using chirality to dictate dimensions and to store chiral information in self-assembled nanotubes in a fully controlled manner is presented. We report a photoresponsive amphiphile that co-assembles with its chiral counterpart to form nanotubes and demonstrate how chirality can be used to effect the formation of either micrometer long, achiral nanotubes or shorter (∼300 nm) chiral nanotubes that are bundled. The nature of these assemblies is studied using a variety of spectroscopic and microscopic techniques and it is shown that the tubes can be disassembled with light, thereby allowing the chiral information to be erased. PMID:28451300

Remote focusing for programmable multi-layer differential multiphoton microscopy

PubMed Central

Hoover, Erich E.; Young, Michael D.; Chandler, Eric V.; Luo, Anding; Field, Jeffrey J.; Sheetz, Kraig E.; Sylvester, Anne W.; Squier, Jeff A.

2010-01-01

We present the application of remote focusing to multiphoton laser scanning microscopy and utilize this technology to demonstrate simultaneous, programmable multi-layer imaging. Remote focusing is used to independently control the axial location of multiple focal planes that can be simultaneously imaged with single element detection. This facilitates volumetric multiphoton imaging in scattering specimens and can be practically scaled to a large number of focal planes. Further, it is demonstrated that the remote focusing control can be synchronized with the lateral scan directions, enabling imaging in orthogonal scan planes. PMID:21326641

β-casein nanovehicles for oral delivery of chemotherapeutic Drug combinations overcoming P-glycoprotein-mediated multidrug resistance in human gastric cancer cells.

PubMed

Bar-Zeev, Maya; Assaraf, Yehuda G; Livney, Yoav D

2016-04-26

Multidrug resistance (MDR) is a primary obstacle to curative cancer therapy. We have previously demonstrated that β-casein (β-CN) micelles (β-CM) can serve as nanovehicles for oral delivery and target-activated release of hydrophobic drugs in the stomach. Herein we introduce a novel nanosystem based on β-CM, to orally deliver a synergistic combination of a chemotherapeutic drug (Paclitaxel) and a P-glycoprotein-specific transport inhibitor (Tariquidar) individually encapsulated within β-CM, for overcoming MDR in gastric cancer. Light microscopy, dynamic light scattering and zeta potential analyses revealed solubilization of these drugs by β-CN, suppressing drug crystallization. Spectrophotometry demonstrated high loading capacity and good encapsulation efficiency, whereas spectrofluorometry revealed high affinity of these drugs to β-CN. In vitro cytotoxicity assays exhibited remarkable synergistic efficacy against human MDR gastric carcinoma cells with P-glycoprotein overexpression. Oral delivery of β-CN - based nanovehicles carrying synergistic drug combinations to the stomach constitutes a novel efficacious therapeutic system that may overcome MDR in gastric cancer.

Antimicrobial activity and mechanism of the human milk-sourced peptide Casein201

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

Zhang, Fan; Department of Endocrinology, Children's Hospital of Nanjing Medical University, Nanjing; Cui, Xianwei

Introduction: Casein201 is one of the human milk sourced peptides that differed significantly in preterm and full-term mothers. This study is designed to demonstrate the biological characteristics, antibacterial activity and mechanisms of Casein201 against common pathogens in neonatal infection. Methodology: The analysis of biological characteristics was done by bioinformatics. Disk diffusion method and flow cytometry were used to detect the antimicrobial activity of Casein201. Killing kinetics of Casein201 was measured using microplate reader. The antimicrobial mechanism of Casein201 was studied by electron microscopy and electrophoresis. Results: Bioinformatics analysis indicates that Casein201 derived from β-casein and showed significant sequence overlap. Antibacterialmore » assays showed Casein201 inhibited the growth of S taphylococcus aureus and Y ersinia enterocolitica. Ultrastructural analyses revealed that the antibacterial activity of Casein201 is through cytoplasmic structures disintegration and bacterial cell envelope alterations but not combination with DNA. Conclusion: We conclude the antimicrobial activity and mechanism of Casein201. Our data demonstrate that Casein201 has potential therapeutic value for the prevention and treatment of pathogens in neonatal infection.« less

Antimicrobial activity and mechanism of the human milk-sourced peptide Casein201.

PubMed

Zhang, Fan; Cui, Xianwei; Fu, Yanrong; Zhang, Jun; Zhou, Yahui; Sun, Yazhou; Wang, Xing; Li, Yun; Liu, Qianqi; Chen, Ting

2017-04-08

Casein201 is one of the human milk sourced peptides that differed significantly in preterm and full-term mothers. This study is designed to demonstrate the biological characteristics, antibacterial activity and mechanisms of Casein201 against common pathogens in neonatal infection. The analysis of biological characteristics was done by bioinformatics. Disk diffusion method and flow cytometry were used to detect the antimicrobial activity of Casein201. Killing kinetics of Casein201 was measured using microplate reader. The antimicrobial mechanism of Casein201 was studied by electron microscopy and electrophoresis. Bioinformatics analysis indicates that Casein201 derived from β-casein and showed significant sequence overlap. Antibacterial assays showed Casein201 inhibited the growth of S taphylococcus aureus and Y ersinia enterocolitica. Ultrastructural analyses revealed that the antibacterial activity of Casein201 is through cytoplasmic structures disintegration and bacterial cell envelope alterations but not combination with DNA. We conclude the antimicrobial activity and mechanism of Casein201. Our data demonstrate that Casein201 has potential therapeutic value for the prevention and treatment of pathogens in neonatal infection. Copyright © 2017 Elsevier Inc. All rights reserved.

A composite chitosan-gelatin bi-layered, biomimetic macroporous scaffold for blood vessel tissue engineering.

PubMed

Badhe, Ravindra V; Bijukumar, Divya; Chejara, Dharmesh R; Mabrouk, Mostafa; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Kondiah, Pierre P D; Pillay, Viness

2017-02-10

A composite chitosan-gelatin macroporous hydrogel-based scaffold with bi-layered tubular architecture was engineered by solvent casting-co-particulate leaching. The scaffold constituted an inner macroporous layer concealed by a non-porous outer layer mimicking the 3D matrix of blood vessels with cellular adhesion and proliferation. The scaffold was evaluated for its morphological, physicochemical, physicomechanical and biodurability properties employing SEM, FTIR, DSC, XRD, porositometry, rheology and texture analysis. The fluid uptake and biodegradation in the presence of lysozymes was also investigated. Cellular attachment and proliferation was analysed using human dermal fibroblasts (HDF-a) seeded onto the scaffold and evaluated by MTT assay, SEM, and confocal microscopy. Results demonstrated that the scaffold had a desirable tensile strength=95.81±11kPa, elongation at break 112.5±13%, porosity 82% and pores between 100 and 230μm, 50% in vitro biodegradation at day 16 and proliferated fibroblasts over 20 days. These results demonstrate that scaffold may be an excellent tubular archetype for blood vessel tissue engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Leakage radiation interference microscopy.

PubMed

Descrovi, Emiliano; Barakat, Elsie; Angelini, Angelo; Munzert, Peter; De Leo, Natascia; Boarino, Luca; Giorgis, Fabrizio; Herzig, Hans Peter

2013-09-01

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

Functional photoacoustic microscopy of pH.

PubMed

Chatni, Muhammad Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P; Maslov, Konstantin I; Wang, Lihong V

2011-10-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.

Functional photoacoustic microscopy of pH

PubMed Central

Chatni, Muhammad Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

2011-01-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy. PMID:22029342

IMART software for correction of motion artifacts in images collected in intravital microscopy

PubMed Central

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

2014-01-01

Intravital microscopy is a uniquely powerful tool, providing the ability to characterize cell and organ physiology in the natural context of the intact, living animal. With the recent development of high-resolution microscopy techniques such as confocal and multiphoton microscopy, intravital microscopy can now characterize structures at subcellular resolution and capture events at sub-second temporal resolution. However, realizing the potential for high resolution requires remarkable stability in the tissue. Whereas the rigid structure of the skull facilitates high-resolution imaging of the brain, organs of the viscera are free to move with respiration and heartbeat, requiring additional apparatus for immobilization. In our experience, these methods are variably effective, so that many studies are compromised by residual motion artifacts. Here we demonstrate the use of IMART, a software tool for removing motion artifacts from intravital microscopy images collected in time series or in three dimensions. PMID:26090271

Advanced spinning disk-TIRF microscopy for faster imaging of the cell interior and the plasma membrane.

PubMed

Zobiak, Bernd; Failla, Antonio Virgilio

2018-03-01

Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Revelation of graphene-Au for direct write deposition and characterization

NASA Astrophysics Data System (ADS)

Bhandari, Shweta; Deepa, Melepurath; Joshi, Amish G.; Saxena, Aditya P.; Srivastava, Avanish K.

2011-06-01

Graphene nanosheets were prepared using a modified Hummer's method, and Au-graphene nanocomposites were fabricated by in situ reduction of a gold salt. The as-produced graphene was characterized by X-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscopy, and high-resolution transmission electron microscopy (HR-TEM). In particular, the HR-TEM demonstrated the layered crystallites of graphene with fringe spacing of about 0.32 nm in individual sheets and the ultrafine facetted structure of about 20 to 50 nm of Au particles in graphene composite. Scanning helium ion microscopy (HIM) technique was employed to demonstrate direct write deposition on graphene by lettering with gaps down to 7 nm within the chamber of the microscope. Bare graphene and graphene-gold nanocomposites were further characterized in terms of their composition and optical and electrical properties.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

In vivo confocal microscopy of human cornea covered with human amniotic membrane.

PubMed

Mimura, Tatsuya; Yamagami, Satoru; Usui, Tomohiko; Honda, Norihiko; Araki, Fumiyuki; Amano, Shiro

2008-01-01

Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22-24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.

Gain-guided soliton fiber laser with high-quality rectangle spectrum for ultrafast time-stretch microscopy.

PubMed

Hu, Song; Yao, Jian; Liu, Meng; Luo, Ai-Ping; Luo, Zhi-Chao; Xu, Wen-Cheng

2016-05-16

The ultrafast time-stretch microscopy has been proposed to enhance the temporal resolution of a microscopy system. The optical source is a key component for ultrafast time-stretch microscopy system. Herein, we reported on the gain-guided soliton fiber laser with high-quality rectangle spectrum for ultrafast time-stretch microscopy. By virtue of the excellent characteristics of the gain-guided soliton, the output power and the 3-dB bandwidth of the stable mode-locked soliton could be up to 3 mW and 33.7 nm with a high-quality rectangle shape, respectively. With the proposed robust optical source, the ultrafast time-stretch microscopy with the 49.6 μm resolution and a scan rate of 11 MHz was achieved without the external optical amplification. The obtained results demonstrated that the gain-guided soliton fiber laser could be used as an alternative high-quality optical source for ultrafast time-stretch microscopy and will introduce some applications in fields such as biology, chemical, and optical sensing.

Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea.

PubMed

Matteini, Paolo; Sbrana, Francesca; Tiribilli, Bruno; Pini, Roberto

2009-07-01

Low-temperature laser welding of the cornea is a technique used to facilitate the closure of corneal cuts. The procedure consists of staining the wound with a chromophore (indocyanine green), followed by continuous wave irradiation with an 810 nm diode laser operated at low power densities (12-16 W/cm(2)), which induces local heating in the 55-65 degrees C range. In this study, we aimed to investigate the ultrastructural modifications in the extracellular matrix following laser welding of corneal wounds by means of atomic force microscopy and transmission electron microscopy. The results evidenced marked disorganization of the normal fibrillar assembly, although collagen appeared not to be denatured under the operating conditions we employed. The mechanism of low-temperature laser welding may be related to some structural modifications of the nonfibrillar extracellular components of the corneal stroma.

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

PubMed

Timmermans, F J; Otto, C

2015-01-01

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

Crystal growth of carbonate apatite using a CaCO3 flux.

PubMed

Suetsugu, Y; Tanaka, J

1999-09-01

Single crystals of carbonate apatite were grown using a CaCO3 flux under an Ar gas pressure of 55 MPa. The crystals obtained were observed by scanning electron microscopy, optical microscopy and X-ray diffraction. Electron probe microanalyses and thermal analyses were performed. CO3 ions in planar triangle form replaced both OH sites and PO4 tetrahedral sites in the apatite structure: in particular, the OH sites were perfectly substituted by CO3 ions using this method.

Surfactant-Templated Mesoporous Metal Oxide Nanowires

DOE PAGES

Luo, Hongmei; Lin, Qianglu; Baber, Stacy; ...

2010-01-01

We demore » monstrate two approaches to prepare mesoporous metal oxide nanowires by surfactant assembly and nanoconfinement via sol-gel or electrochemical deposition. For example, mesoporous Ta 2 O 5 and zeolite nanowires are prepared by block copolymer Pluronic 123-templated sol-gel method, and mesoporous ZnO nanowires are prepared by electrodeposition in presence of anionic surfactant sodium dodecyl sulfate (SDS) surfactant, in porous membranes. The morphologies of porous nanowires are studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses.« less

Copper Decoration of Carbon Nanotubes and High Resolution Electron Microscopy

NASA Astrophysics Data System (ADS)

Probst, Camille

A new process of decorating carbon nanotubes with copper was developed for the fabrication of nanocomposite aluminum-nanotubes. The process consists of three stages: oxidation, activation and electroless copper plating on the nanotubes. The oxidation step was required to create chemical function on the nanotubes, essential for the activation step. Then, catalytic nanoparticles of tin-palladium were deposited on the tubes. Finally, during the electroless copper plating, copper particles with a size between 20 and 60 nm were uniformly deposited on the nanotubes surface. The reproducibility of the process was shown by using another type of carbon nanotube. The fabrication of nanocomposites aluminum-nanotubes was tested by aluminum vacuum infiltration. Although the infiltration of carbon nanotubes did not produce the expected results, an interesting electron microscopy sample was discovered during the process development: the activated carbon nanotubes. Secondly, scanning transmitted electron microscopy (STEM) imaging in SEM was analysed. The images were obtained with a new detector on the field emission scanning electron microscope (Hitachi S-4700). Various parameters were analysed with the use of two different samples: the activated carbon nanotubes (previously obtained) and gold-palladium nanodeposits. Influences of working distance, accelerating voltage or sample used on the spatial resolution of images obtained with SMART (Scanning Microscope Assessment and Resolution Testing) were analysed. An optimum working distance for the best spatial resolution related to the sample analysed was found for the imaging in STEM mode. Finally, relation between probe size and spatial resolution of backscattered electrons (BSE) images was studied. An image synthesis method was developed to generate the BSE images from backscattered electrons coefficients obtained with CASINO software. Spatial resolution of images was determined using SMART. The analysis shown that using a probe size smaller than the size of the observed object (sample features) does not improve the spatial resolution. In addition, the effects of the accelerating voltage, the current intensity and the sample geometry and composition were analysed.

Quantitative light and scanning electron microscopy of ferret sperm.

PubMed

Van der Horst, G; Curry, P T; Kitchin, R M; Burgess, W; Thorne, E T; Kwiatkowski, D; Parker, M; Atherton, R W

1991-11-01

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

PubMed

Kraus, Max-Joseph; Seifert, Jan; Strasser, Erwin F; Gawaz, Meinrad; Schäffer, Tilman E; Rheinlaender, Johannes

2016-09-01

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

Imaging of matrix-disorder in normal and pathological human dermis using nonlinear optical microscopy

NASA Astrophysics Data System (ADS)

Zhuo, Shuangmu; Chen, Jianxin; Xie, Shusen; Zheng, Liqin; Jiang, Xingshan

2009-11-01

In dermis, collagen and elastin are important structural proteins of extracellular maxtrix. The matrix-disorder is associated with various physiologic processes, such as localized scleroderma, anetoderma, photoaging. In this work, we demonstrate the capability of nonlinear optical microscopy in imaging structural proteins in normal and pathological human dermis.

Topographical and Chemical Imaging of a Phase Separated Polymer Using a Combined Atomic Force Microscopy/Infrared Spectroscopy/Mass Spectrometry Platform

DOE PAGES

Tai, Tamin; Karácsony, Orsolya; Bocharova, Vera; ...

2016-02-18

This article describes how the use of a hybrid atomic force microscopy/infrared spectroscopy/mass spectrometry imaging platform was demonstrated for the acquisition and correlation of nanoscale sample surface topography and chemical images based on infrared spectroscopy and mass spectrometry.

Brillouin microscopy: assessing ocular tissue biomechanics.

PubMed

Yun, Seok Hyun; Chernyak, Dimitri

2018-07-01

Assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. Many researchers and clinicians have identified corneal biomechanics as source of variability in refractive procedures and one of the main factors in keratoconus. However, it has been difficult to accurately characterize corneal biomechanics in patients. The recent development of Brillouin light scattering microscopy heightens the promise of bringing biomechanics into the clinic. The aim of this review is to overview the progress and discuss prospective applications of this new technology. Brillouin microscopy uses a low-power near-infrared laser beam to determine longitudinal modulus or mechanical compressibility of tissue by analyzing the return signal spectrum. Human clinical studies have demonstrated significant difference in the elastic properties of normal corneas versus corneas diagnosed with mild and severe keratoconus. Clinical data have also shown biomechanical changes after corneal cross-linking treatment of keratoconus patients. Brillouin measurements of the crystalline lens and sclera have also been demonstrated. Brillouin microscopy is a promising technology under commercial development at present. The technique enables physicians to characterize the biomechanical properties of ocular tissues.

Magnetic removal of Entamoeba cysts from water using chitosan oligosaccharide-coated iron oxide nanoparticles

PubMed Central

Shukla, Sudeep; Arora, Vikas; Jadaun, Alka; Kumar, Jitender; Singh, Nishant; Jain, Vinod Kumar

2015-01-01

Amebiasis, a major health problem in developing countries, is the second most common cause of death due to parasitic infection. Amebiasis is usually transmitted by the ingestion of Entamoeba histolytica cysts through oral–fecal route. Herein, we report on the use of chitosan oligosaccharide-functionalized iron oxide nanoparticles for efficient capture and removal of pathogenic protozoan cysts under the influence of an external magnetic field. These nanoparticles were synthesized through a chemical synthesis process. The synthesized particles were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and zeta potential analysis. The particles were found to be well dispersed and uniform in size. The capture and removal of pathogenic cysts were demonstrated by fluorescent microscopy, transmission electron microscopy, and scanning electron microscopy (SEM). Three-dimensional modeling of various biochemical components of cyst walls, and thereafter, flexible docking studies demonstrate the probable interaction mechanism of nanoparticles with various components of E. histolytica cyst walls. Results of the present study suggest that E. histolytica cysts can be efficiently captured and removed from contaminated aqueous systems through the application of synthesized nanoparticles. PMID:26261417

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

PubMed

Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

2015-01-01

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

PubMed

Richards, Thomas A; Leonard, Guy; Mahé, Frédéric; Del Campo, Javier; Romac, Sarah; Jones, Meredith D M; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-11-22

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. © 2015 The Authors.

Molecular diversity and distribution of marine fungi across 130 European environmental samples

PubMed Central

Richards, Thomas A.; Leonard, Guy; Mahé, Frédéric; del Campo, Javier; Romac, Sarah; Jones, Meredith D. M.; Maguire, Finlay; Dunthorn, Micah; De Vargas, Colomban; Massana, Ramon; Chambouvet, Aurélie

2015-01-01

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal ‘OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments. PMID:26582030

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy.

PubMed

Gianoncelli, A; Vaccari, L; Kourousias, G; Cassese, D; Bedolla, D E; Kenig, S; Storici, P; Lazzarino, M; Kiskinova, M

2015-05-14

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy

PubMed Central

Gianoncelli, A.; Vaccari, L.; Kourousias, G.; Cassese, D.; Bedolla, D. E.; Kenig, S.; Storici, P.; Lazzarino, M.; Kiskinova, M.

2015-01-01

Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies. PMID:25974639

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

Cooke, Gary A.; Pestovich, John A.; Huber, Heinz J.

This report presents the results for solid phase characterization (SPC) of solid samples removed from tank 241-C-108 (C-108) on August 12-13,2012, using the off-riser sampler. Samples were received at the 222-S Laboratory on August 13 and were described and photographed. The SPC analyses that were performed include scanning electron microscopy (SEM) using the ASPEX(R)l scanning electron microscope, X-ray diffraction (XRD) using the Rigaku(R) 2 MiniFlex X-ray diffractometer, and polarized light microscopy (PLM) using the Nikon(R) 3 Eclipse Pol optical microscope. The SEM is equipped with an energy dispersive X-ray spectrometer (EDS) to provide chemical information. Gary A. Cooke conducted themore » SEM analysis, John A. Pestovich performed the XRD analysis, and Dr. Heinz J. Huber performed the PLM examination. The results of these analyses are presented here.« less

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

Zhao, Donghui; Key Lab of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi, Changning, Shanghai 200050; Zhu, Yingchun, E-mail: yzhu@mail.sic.ac.cn

In this article, the polymorph selection of calcium carbonate has been successfully achieved in water-soluble carboxymethyl chitosan aqueous solution at different temperatures (25-95 {sup o}C). Vaterite is formed in carboxymethyl chitosan solution 25 {sup o}C accompanied with trace of calcite, whereas pure aragonite is obtained at 95 {sup o}C. Scanning electron microscopy and transmission electron microscopy analyses show that the products are formed from the recrystallization of nanometer crystallites. Thermodynamic and kinetic analyses reveal that the polymorph of calcium carbonate is controlled and selected by kinetics in various temperatures. As a heterogeneous nucleator and stabilizing agent, carboxymethyl chitosan changes themore » nucleation and growth of calcium carbonate from thermodynamic into kinetic control. Under kinetic limitation, the reaction rate of aragonite increases along with the elevating of temperature and surpasses the rate of vaterite above 327 K.« less

Effects of mild ozonisation on gene expression and nuclear domains organization in vitro.

PubMed

Scassellati, C; Costanzo, M; Cisterna, B; Nodari, A; Galiè, M; Cattaneo, A; Covi, V; Tabaracci, G; Bonvicini, C; Malatesta, M

2017-10-01

In the last two decades, the use of ozone (O 3 ) as a complementary medical approach has progressively been increasing; however, its application is still limited due to the numerous doubts about its possible toxicity, despite the low concentrations used in therapy. For an appropriate and safe clinical application of a potentially toxic agent such as O 3 , it is crucial to elucidate the cellular response to its administration. Molecular analyses and transmission electron microscopy were here combined to investigate in vitro the effects of O 3 administration on transcriptional activity and nuclear domains organization of cultured SH-SY5Y neuronal cells; low O 3 concentrations were used as those currently administered in clinical practice. Mild ozonisation did not affect cell proliferation or death, while molecular analyses showed an O 3 -induced modulation of some genes involved in the cell response to stress (HMOX1, ERCC4, CDKN1A) and in the transcription machinery (CTDSP1). Ultrastructural cytochemistry after experiments of bromouridine incorporation consistently demonstrated an increased transcriptional rate at both the nucleoplasmic (mRNA) and the nucleolar (rRNA) level. No ultrastructural alteration of nuclear domains was observed. Our molecular, ultrastructural and cytochemical data demonstrate that a mild toxic stimulus such as mild ozonisation stimulate cell protective pathways and nuclear transcription, without altering cell viability. This could possibly account for the positive effects observed in ozone-treated patients. Copyright © 2017 Elsevier Ltd. All rights reserved.

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

PubMed

Erlandsen, S L; Sherlock, L A; Bemrick, W J

1990-04-01

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Mll5 Is Required for Normal Spermatogenesis

PubMed Central

Yap, Damian B.; Walker, David C.; Prentice, Leah M.; McKinney, Steven; Turashvili, Gulisa; Mooslehner-Allen, Katrin; de Algara, Teresa Ruiz; Fee, John; de Tassigny, Xavier d'Anglemont; Colledge, William H.; Aparicio, Samuel

2011-01-01

Background Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis. Methodology/Principal Findings Mll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated. Conclusions/Significance Our data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects. PMID:22069496

Photosynthetic and ultrastructural responses of Ulva australis to Zn stress.

PubMed

Farias, D R; Schmidt, E; Simioni, C; Bouzon, Z L; Hurd, C L; Eriksen, R S; Macleod, C K

2017-12-01

This research evaluated the effect of zinc (Zn) on the ultrastructure and the photosynthetic efficiency of a common green alga. Ulva australis was grown in the laboratory for 7days under a range of different Zn concentrations (0, 25, 50 and 100μgL -1 ). Growth rate (Gr), photosynthetic efficiency (Fv/Fm and ETRmax), photosynthetic pigments, and metal accumulation were measured. Samples of 1mm length were taken to analyse the effect of Zn on the ultrastructure using transmission electron microscopy (TEM) and cytochemical responses (TB-O and PAS) were evaluated by light microscopy (LM). There were no significant differences in the growth rate, Fv/Fm, ETRmax and the photosynthetic pigments chlorophyll a, chlorophyll b and carotenoids (p>0.05) after 7days of Zn exposure. However, TEM revealed cytoplasm retraction, compression of cellulose fibrils, dissembled thylakoids and electron-dense bodies suggesting ultrastructural impacts from metal exposure and accumulation. Cytological analysis demonstrated that Zn affected U. australis cells at the three concentrations tested. The main effect was cytoplasm retraction and a decrease on the amount of starch granules, following exposure at 25μgL -1 and 50μgL -1 of Zn. We conclude that concentrations of Zn assessed in U. australis in this research has a short-term cellular effect as revealed by TEM and cytological analysis, demonstrating the importance of measuring a broad suite of endpoints to better understand species responses to environmentally relevant concentrations of Zn. However, U. australis was able to physiologically tolerate adverse conditions, since there was no effect on the photosynthetic performance and growth. Copyright © 2017 Elsevier Ltd. All rights reserved.

A large-sized bubbling appearance of the glomerular basement membrane in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis.

PubMed

Suga, Norihiro; Miura, Naoto; Uemura, Yuko; Nakamura, Toshinobu; Morita, Hiroyuki; Banno, Shogo; Imai, Hirokazu

2011-12-01

We report an unusual pathological finding, a large-sized bubbling appearance of the glomerular basement membrane (GBM), in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis. The first renal biopsy specimen from 10 years ago, when systemic lupus erythematosus was diagnosed, demonstrated mild mesangial proliferation and subepithelial deposits (WHO classification: III + V). Light microscopy of the current biopsy using periodic acid methenamine silver (PAMS) stain demonstrated a large-sized bubbling appearance of the GBM; however, very weak immunoglobulin and complement deposition was observed in immunofluorescence studies. Routine electron microscopy demonstrated partial subendothelial expansion with electron-lucent materials, but no electron-dense deposits or amyloid fibrils. Electron microscopy with PAMS stain revealed electron-lucent endothelial scalloping, including some cellular components and microspheres in the GBM; however, it is not clear if these materials are derived from endothelial cells. One possibility is that these unique findings represent a recovery phase of lupus membranous nephritis; another is that these findings correspond to a new disease entity.

Fibronectin non-amyloid glomerulopathy.

PubMed

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2009-11-20

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Fibronectin non-amyloid glomerulopathy

PubMed Central

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2010-01-01

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 µmol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology. PMID:20126589

Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

PubMed

Barlow, Andrew L; Macleod, Alasdair; Noppen, Samuel; Sanderson, Jeremy; Guérin, Christopher J

2010-12-01

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

In vitro behavior of human mesenchymal stem cells on poly(N-isopropylacrylamide) based biointerfaces obtained by matrix assisted pulsed laser evaporation

NASA Astrophysics Data System (ADS)

Icriverzi, Madalina; Rusen, Laurentiu; Sima, Livia Elena; Moldovan, Antoniu; Brajnicov, Simona; Bonciu, Anca; Mihailescu, Natalia; Dinescu, Maria; Cimpean, Anisoara; Roseanu, Anca; Dinca, Valentina

2018-05-01

The use of smart coatings with tunable characteristics in bioengineering fields is directly correlated with the surface chemical and topographical properties, the method of preparation, and also with the type of cells implied for the specific application. In this work, a versatile surface modification technique based on the use of lasers (Matrix-Assisted Pulsed Laser Evaporation (MAPLE)) was used to yield poly(N-isopropylacrylamide) (pNIPAM) and its derivatives (amine, azide and amide terminated pNIPAM) functional and termoresponsive thin films. Surface properties of pNIPAM and its derivative films such as morphology, roughness and hydrophobic/hydrophilic character, as well as the thermoresponsive capacity were investigated by atomic force microscopy and contact angle measurements. The chemical characteristics of the pNIPAM based thin films were analysed by Fourier Transform Infrared Spectroscopy (FTIR). The chemical functionality was kept for all the samples obtained by MAPLE and the thermoresponse was demonstrated by the change in the contact angle and thickness values when the temperature was shifted from 37 °C to 24 °C for all the materials tested, with a smaller change for maleimide terminated pNIPAM. Biological assays performed in vitro (fluorescence microscopy and Scanning Electron Microscopy (SEM)) confirmed the conditioning of the early mesenchymal stem cell (MSC) growth by specific chemistry of the coatings. The cell imaging analysis revealed no cytotoxic effect of pNIPAM surfaces irrespective of type of functionalization. An increased proliferation rate of the cells grown on pNIPAM-azide surfaces and a lower cell density on pNIPAM-maleimide surfaces compared to the pNIPAM surfaces was observed, which can direct their use to potential surfaces in regenerative medicine approaches.

Effect of an exfoliating skincare regimen on the numbers of epithelial squames on the skin of operating theatre staff, studied by surface microscopy.

PubMed

Wernham, A G; Cain, O L; Thomas, A M

2018-03-23

The shedding of epithelial squames (skin scales) by staff in operating theatre air is an important source of deep infection following joint replacement surgery. This is a serious complication, resulting in significant morbidity for the patient and substantial cost implications for healthcare systems. Much effort has been put into providing clean air in operating theatres, yet little attention has been given to reducing the shedding of surface skin scales at source. To develop a novel method for calculating surface skin scale density using surface microscopy, and to use it to evaluate the effect of a skincare regimen on operating theatre staff. Surface microscopy with Z-stacked imaging was used to visualize the effect of a skincare regimen involving three stages: washing with soap; exfoliation; and application of emollient. A USB microscope was then used in a field study to take images of the skin of operating theatre staff who applied the regimen to one lower limb the night before testing. The other limb was used as a control. Two blinded assessors analysed scale density. Z-stack images from the surface microscope enabled observations of the skincare regimen. The USB microscope also provided adequate images that enabled assessment of skin scale density. In the operating theatre staff, a 72.1% reduction in visible skin scales was observed following application of the skincare regimen. Further work is required to demonstrate how this effect correlates with dispersion of skin particles in a cleanroom, and subsequently in live operating theatre studies. Copyright © 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure.

PubMed

Abadie, S; Jardet, C; Colombelli, J; Chaput, B; David, A; Grolleau, J-L; Bedos, P; Lobjois, V; Descargues, P; Rouquette, J

2018-05-01

Human skin is composed of the superimposition of tissue layers of various thicknesses and components. Histological staining of skin sections is the benchmark approach to analyse the organization and integrity of human skin biopsies; however, this approach does not allow 3D tissue visualization. Alternatively, confocal or two-photon microscopy is an effective approach to perform fluorescent-based 3D imaging. However, owing to light scattering, these methods display limited light penetration in depth. The objectives of this study were therefore to combine optical clearing and light-sheet fluorescence microscopy (LSFM) to perform in-depth optical sectioning of 5 mm-thick human skin biopsies and generate 3D images of entire human skin biopsies. A benzyl alcohol and benzyl benzoate solution was used to successfully optically clear entire formalin fixed human skin biopsies, making them transparent. In-depth optical sectioning was performed with LSFM on the basis of tissue-autofluorescence observations. 3D image analysis of optical sections generated with LSFM was performed by using the Amira ® software. This new approach allowed us to observe in situ the different layers and compartments of human skin, such as the stratum corneum, the dermis and epidermal appendages. With this approach, we easily performed 3D reconstruction to visualise an entire human skin biopsy. Finally, we demonstrated that this method is useful to visualise and quantify histological anomalies, such as epidermal hyperplasia. The combination of optical clearing and LSFM has new applications in dermatology and dermatological research by allowing 3D visualization and analysis of whole human skin biopsies. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Investigation of quartz grain surface textures by atomic force microscopy for forensic analysis.

PubMed

Konopinski, D I; Hudziak, S; Morgan, R M; Bull, P A; Kenyon, A J

2012-11-30

This paper presents a study of quartz sand grain surface textures using atomic force microscopy (AFM) to image the surface. Until now scanning electron microscopy (SEM) has provided the primary technique used in the forensic surface texture analysis of quartz sand grains as a means of establishing the provenance of the grains for forensic reconstructions. The ability to independently corroborate the grain type classifications is desirable and provides additional weight to the findings of SEM analysis of the textures of quartz grains identified in forensic soil/sediment samples. AFM offers a quantitative means of analysis that complements SEM examination, and is a non-destructive technique that requires no sample preparation prior to scanning. It therefore has great potential to be used for forensic analysis where sample preservation is highly valuable. By taking quantitative topography scans, it is possible to produce 3D representations of microscopic surface textures and diagnostic features for examination. Furthermore, various empirical measures can be obtained from analysing the topography scans, including arithmetic average roughness, root-mean-square surface roughness, skewness, kurtosis, and multiple gaussian fits to height distributions. These empirical measures, combined with qualitative examination of the surfaces can help to discriminate between grain types and provide independent analysis that can corroborate the morphological grain typing based on the surface textures assigned using SEM. Furthermore, the findings from this study also demonstrate that quartz sand grain surfaces exhibit a statistically self-similar fractal nature that remains unchanged across scales. This indicates the potential for a further quantitative measure that could be utilised in the discrimination of quartz grains based on their provenance for forensic investigations. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The fate of radiation induced giant-nucleated cells of human skin fibroblasts

NASA Astrophysics Data System (ADS)

Almahwasi, A. A.; Jeynes, J. C.; Bradley, D. A.; Regan, P. H.

2017-11-01

Radiation-induced giant-nucleated cells (GCs) have been observed to occur within survivors of irradiated cancerous and within healthy cells, both in vivo and in vitro. The expression of such morphological alterations is associated with genomic instability. This study was designed to investigate the fate of GCs induced in a normal human fibroblast cell line (AG1522) after exposure to 0.2, 1 or 2 Gy of X-ray or proton irradiation. The total of 79 individual AG1522 GCs present at 7, 14 or 21 days after each dose point were analysed from fluorescence microscopy images captured over approximately 120 h. The GCs were identified at the beginning of the observation period for each time point post-irradiation and the area of the cell nucleus was measured (μm2) using a cell-recognition MATLAB code. The results demonstrate that the majority of GCs had undergone a prolonged mitotic arrest, which might be an indication of the survival strategy. The live cell microscopy confirms that a giant-nucleated cell formed 14 days after exposure to 0.2 Gy of proton irradiation was divided into two asymmetrical normal-sized cells. These results suggest that a small fraction of GCs can proliferate and form progeny. Some of GCs had disappeared from the microscopy fields. The rate of their loss was decreased as the dose increased but there remains the potential for them to have progeny that could continue to proliferate, ultimately contributing to development of cancer risk. This important method to access delayed effects in normal tissues could act as a potential radioprotective assay for a dose-limiting parameter when applying radiotherapy. These results might have important implications in evaluating risk estimates for patients during radiation therapy treatment.

Association between dermoscopic and reflectance confocal microscopy features of cutaneous melanoma with BRAF mutational status.

PubMed

Bombonato, C; Ribero, S; Pozzobon, F C; Puig-Butille, J A; Badenas, C; Carrera, C; Malvehy, J; Moscarella, E; Lallas, A; Piana, S; Puig, S; Argenziano, G; Longo, C

2017-04-01

Melanomas harbouring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy. BRAF mutational status is crucial for the management of metastatic melanoma. To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumours has also been analysed for the presence of possible confocal features that might be linked with BRAF status. Retrospectively acquired dermoscopic and confocal images of patients with melanoma in tertiary referral academic centres: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskal-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. Dermoscopically, the presence of irregular peripheral streaks and ulceration were positive predictors of BRAF-mutated melanomas with a statistically significance value, while dotted vessels were more represented in wild-type melanomas. None of the evaluated reflectance confocal microscopy features were correlated with genetic profiling. Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF-mutated melanoma, while dotted vessels are suggestive for wild-type melanoma. © 2016 European Academy of Dermatology and Venereology.

Changes in biooxidation mechanism and transient biofilm characteristics by As(V) during arsenopyrite colonization with Acidithiobacillus thiooxidans.

PubMed

Ramírez-Aldaba, Hugo; Vázquez-Arenas, Jorge; Sosa-Rodríguez, Fabiola S; Valdez-Pérez, Donato; Ruiz-Baca, Estela; Trejo-Córdoba, Gabriel; Escobedo-Bretado, Miguel A; Lartundo-Rojas, Luis; Ponce-Peña, Patricia; Lara, René H

2018-06-01

Chemical and surface analyses are carried out using Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM-EDS), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), glow discharge spectroscopy (GDS) and extracellular surface protein quantification to thoroughly investigate the effect of supplementary As(V) during biooxidation of arsenopyrite by Acidithiobacillus thiooxidans. It is revealed that arsenic can enhance bacterial reactions during bioleaching, which can strongly influence its mobility. Biofilms occur as compact-flattened microcolonies, being progressively covered by a significant amount of secondary compounds (S n 2- , S 0 , pyrite-like). Biooxidation mechanism is modified in the presence of supplementary As(V), as indicated by spectroscopic and microscopic studies. GDS confirms significant variations between abiotic control and biooxidized arsenopyrite in terms of surface reactivity and amount of secondary compounds with and without As(V) (i.e. 6 μm depth). CLSM and protein analyses indicate a rapid modification in biofilm from hydrophilic to hydrophobic character (i.e. 1-12 h), in spite of the decrease in extracellular surface proteins in the presence of supplementary As(V) (i.e. stressed biofilms).

Case management of malaria in Swaziland, 2011-2015: on track for elimination?

PubMed

Dlamini, S V; Kosgei, R J; Mkhonta, N; Zulu, Z; Makadzange, K; Zhou, S; Owiti, P; Sikhondze, W; Namboze, J; Reid, A; Kunene, S

2018-04-25

Objective: To assess adherence to malaria diagnosis and treatment guidelines (2010 and 2014) in all health care facilities in Swaziland between 2011 and 2015. Methods: This was a cross-sectional descriptive study involving all health care facilities that diagnosed and managed malaria cases in Swaziland. Patients' age, sex, diagnosis method and type of treatment were analysed. Results: Of 1981 records for severe and uncomplicated malaria analysed, 56% of cases were uncomplicated and 14% had severe malaria. The type of malaria was not recorded for 30% of cases. Approximately 71% of cases were confirmed by rapid diagnostic tests (RDT) alone, 3% by microscopy alone and 26% by both RDT and microscopy. Of the uncomplicated cases, 93% were treated with artemether-lumefantrine (AL) alone, 5% with quinine alone and 2% with AL and quinine. Amongst the severe cases, 11% were treated with AL alone, 44% with quinine alone and 45% with AL and quinine. For severe malaria, clinics and health centres prescribed AL alone more often than hospitals (respectively 13%, 12% and 4%, P = 0.03). Conclusion: RDTs and/or microscopy results are used at all facilities to inform treatment. Poor recording of malaria type causes difficulties in assessing the prescription of antimalarial drugs.

Developing single-laser sources for multimodal coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Pegoraro, Adrian Frank

Coherent anti-Stokes Raman scattering (CARS) microscopy has developed rapidly and is opening the door to new types of experiments. This work describes the development of new laser sources for CARS microscopy and their use for different applications. It is specifically focused on multimodal nonlinear optical microscopy—the simultaneous combination of different imaging techniques. This allows us to address a diverse range of applications, such as the study of biomaterials, fluid inclusions, atherosclerosis, hepatitis C infection in cells, and ice formation in cells. For these applications new laser sources are developed that allow for practical multimodal imaging. For example, it is shown that using a single Ti:sapphire oscillator with a photonic crystal fiber, it is possible to develop a versatile multimodal imaging system using optimally chirped laser pulses. This system can perform simultaneous two photon excited fluorescence, second harmonic generation, and CARS microscopy. The versatility of the system is further demonstrated by showing that it is possible to probe different Raman modes using CARS microscopy simply by changing a time delay between the excitation beams. Using optimally chirped pulses also enables further simplification of the laser system required by using a single fiber laser combined with nonlinear optical fibers to perform effective multimodal imaging. While these sources are useful for practical multimodal imaging, it is believed that for further improvements in CARS microscopy sensitivity, new excitation schemes are necessary. This has led to the design of a new, high power, extended cavity oscillator that should be capable of implementing new excitation schemes for CARS microscopy as well as other techniques. Our interest in multimodal imaging has led us to other areas of research as well. For example, a fiber-coupling scheme for signal collection in the forward direction is demonstrated that allows for fluorescence lifetime imaging without significant temporal distortion. Also highlighted is an imaging artifact that is unique to CARS microscopy that can alter image interpretation, especially when using multimodal imaging. By combining expertise in nonlinear optics, laser development, fiber optics, and microscopy, we have developed systems and techniques that will be of benefit for multimodal CARS microscopy.

Reflection imaging of China ink-perfused brain vasculature using confocal laser-scanning microscopy after clarification of brain tissue by the Spalteholz method.

PubMed

Gutierre, R C; Vannucci Campos, D; Mortara, R A; Coppi, A A; Arida, R M

2017-04-01

Confocal laser-scanning microscopy is a useful tool for visualizing neurons and glia in transparent preparations of brain tissue from laboratory animals. Currently, imaging capillaries and venules in transparent brain tissues requires the use of fluorescent proteins. Here, we show that vessels can be imaged by confocal laser-scanning microscopy in transparent cortical, hippocampal and cerebellar preparations after clarification of China ink-injected specimens by the Spalteholz method. This method may be suitable for global, three-dimensional, quantitative analyses of vessels, including stereological estimations of total volume and length and of surface area of vessels, which constitute indirect approaches to investigate angiogenesis. © 2017 Anatomical Society.

Changes in resistant starch from two banana cultivars during postharvest storage.

PubMed

Wang, Juan; Tang, Xue Juan; Chen, Ping Sheng; Huang, Hui Hua

2014-08-01

Banana resistant starch samples were extracted and isolated from two banana cultivars (Musa AAA group, Cavendish subgroup and Musa ABB group, Pisang Awak subgroup) at seven ripening stages during postharvest storage. The structures of the resistant starch samples were analysed by light microscopy, polarising microscopy, scanning electron microscopy, X-ray diffraction, and infrared spectroscopy. Physicochemical properties (e.g., water-holding capacity, solubility, swelling power, transparency, starch-iodine absorption spectrum, and Brabender microviscoamylograph profile) were determined. The results revealed significant differences in microstructure and physicochemical characteristics among the banana resistant starch samples during different ripening stages. The results of this study provide valuable information for the potential applications of banana resistant starches. Copyright © 2014 Elsevier Ltd. All rights reserved.

Special issue on high-resolution optical imaging

NASA Astrophysics Data System (ADS)

Smith, Peter J. S.; Davis, Ilan; Galbraith, Catherine G.; Stemmer, Andreas

2013-09-01

The pace of development in the field of advanced microscopy is truly breath-taking, and is leading to major breakthroughs in our understanding of molecular machines and cell function. This special issue of Journal of Optics draws attention to a number of interesting approaches, ranging from fluorescence and imaging of unlabelled cells, to computational methods, all of which are describing the ever increasing detail of the dynamic behaviour of molecules in the living cell. This is a field which traditionally, and currently, demonstrates a marvellous interplay between the disciplines of physics, chemistry and biology, where apparent boundaries to resolution dissolve and living cells are viewed in ever more clarity. It is fertile ground for those interested in optics and non-conventional imaging to contribute high-impact outputs in the fields of cell biology and biomedicine. The series of articles presented here has been selected to demonstrate this interdisciplinarity and to encourage all those with a background in the physical sciences to 'dip their toes' into the exciting and dynamic discoveries surrounding cell function. Although single molecule super-resolution microscopy is commercially available, specimen preparation and interpretation of single molecule data remain a major challenge for scientists wanting to adopt the techniques. The paper by Allen and Davidson [1] provides a much needed detailed introduction to the practical aspects of stochastic optical reconstruction microscopy, including sample preparation, image acquisition and image analysis, as well as a brief description of the different variants of single molecule localization microscopy. Since super-resolution microscopy is no longer restricted to three-dimensional imaging of fixed samples, the review by Fiolka [2] is a timely introduction to techniques that have been successfully applied to four-dimensional live cell super-resolution microscopy. The combination of multiple high-resolution techniques, such as the combination of light sheet and structured illumination microscopy (SIM), which efficiently utilize photon budget and avoid illuminating regions of the specimen not currently being imaged, hold the greatest promise for future biological applications. Therefore, the combined setup for SIM and single molecule localization microscopy (SMLM) described by Rossberger et al [3] will be very helpful and stimulating to advanced microscopists in further modifying their setups. The SIM image helps in identifying artefacts in SMLM reconstruction, e.g. when two active fluorophores are close together and get rejected as 'out-of-focus'. This combined setup is another way to facilitate imaging live samples. The article by Thomas et al [4] presents another advance for biological super-resolution imaging with a new approach to reconstruct optically sectioned images using structured illumination. The method produces images with higher spatial resolution and greater signal to noise compared to existing approaches. This algorithm demonstrates great promise for reconstructing biological images where the signal intensities are inherently lower. Shevchuk et al [5] present a non-optic near field approach to imaging with a review of scanning ion-conductance microscopy. This is a powerful alternative approach for examining the surface dynamics of living cells including exo and endocytosis, unlabelled, and at the level of the single event. Here they present the first data on combining this approach with fluorescence confocal microscopy—adding that extra dimension. Different approaches to label-free live cell imaging are presented in the papers by Patel et al [6], Mehta and Oldenbourg [7], as well as Rogers and Zheludev [8]. All three papers bring home the excitement of looking at live cell dynamics without reporters—Patel et al [6] review both the potential of coherent anti-Stokes Raman scattering and biological applications, where specific biomolecules are detected on the basis of their biophysical properties. Polarized light microscopy as presented by Mehta and Oldenbourg [7], describe a novel implementation of this technology to detect dichroism, and demonstrate beautifully its use in imaging unlabelled microtubules, mitochondria and lipid droplets. Sub-wavelength light focusing provides another avenue to super-resolution, and this is presented by Rogers and Zheludev [8]. Speculating on further improvements, these authors expect a resolution of 0.15λ. To date, the method has not been applied to low contrast, squishy and motile biotargets, but is included here for the clear potential to drive label-free imaging in new directions. A similar logic lies behind the inclusion of Parsons et al [9] where ultraviolet coherent diffractive imaging is further developed. These authors have demonstrated a shrink-wrap technique which reduces the integration time by a factor of 5, bringing closer the time when we have lab based imaging systems based on extreme ultraviolet and soft x-ray sources using sophisticated phase retrieval algorithms. Real biological specimens have spatially varying refractive indices that inevitably lead to aberrations and image distortions. Global refractive index matching of the embedding medium has been an historic solution, but unfortunately is not practical for live cell imaging. Adaptive optics appears an attractive solution and Simmonds and Booth [10] demonstrate the theoretical benefits of applying several adaptive optical elements, placed in different conjugate planes, to create a kind of 'inverse specimen' that unwarps phase distortions of the sample—but these have yet to be tested on real specimens. A difficulty in single molecule localization microscopy has been the determination of whether or not two molecules are colocalized. Kim et al [11] present a method for correcting bleed-through during multi-colour, single molecule localization microscopy. Such methods are welcome standards when trying to quantifiably interpret how close two molecules actually are. Rees et al [12] provide an invaluable overview of key image processing steps in localization microscopy. This paper is an excellent starting point for anyone implementing localization algorithms and the Matlab software provided will be invaluable; a strong paper on which to conclude our overview of the excellent articles brought together in this issue. One aspect brought home in several of these articles is the volume of data now being collected by high resolution live cell imaging. Data processing and image reconstruction will continue to be pressure points in the further development of instrumentation and analyses. We would hope that the series of papers presented here will motivate software engineers, optical physicists and biologists to contribute to the further development of this exciting field. References [1] Allen J R et al 2013 J. Opt. 15 094001 [2] Fiolka R et al 2013 J. Opt. 15 094002 [3] Rossberger S et al 2013 J. Opt. 15 094003 [4] Thomas B et al 2013 J. Opt. 15 094004 [5] Shevchuk A et al 2013 J. Opt. 15 094005 [6] Patel I et al 2013 J. Opt. 15 094006 [7] Mehta S B et al 2013 J. Opt. 15 094007 [8] Rogers E T F et al 2013 J. Opt. 15 094008 [9] Parsons A D et al 2013 J. Opt. 15 094009 [10] Simmonds R et al 2013 J. Opt. 15 094010 [11] Kim D et al 2013 J. Opt. 15 094011 [12] Rees E J et al 2013 J. Opt. 15 094012

Local carrier distribution imaging on few-layer MoS2 exfoliated on SiO2 by scanning nonlinear dielectric microscopy

NASA Astrophysics Data System (ADS)

Yamasue, Kohei; Cho, Yasuo

2018-06-01

We demonstrate that scanning nonlinear dielectric microscopy (SNDM) can be used for the nanoscale characterization of dominant carrier distribution on atomically thin MoS2 mechanically exfoliated on SiO2. For stable imaging without damaging microscopy tips and samples, SNDM was combined with peak-force tapping mode atomic force microscopy. The identification of dominant carriers and their spatial distribution becomes possible even for single and few-layer MoS2 on SiO2 using the proposed method allowing differential capacitance (dC/dV) imaging. We can expect that SNDM can also be applied to the evaluation of other two-dimensional semiconductors and devices.

Demonstration of transmission high energy electron microscopy

DOE PAGES

Merrill, F. E.; Goett, J.; Gibbs, J. W.; ...

2018-04-06

High energy electrons have been used to investigate an extension of transmission electron microscopy. This technique, transmission high energy electron microscopy (THEEM), provides two additional capabilities to electron microscopy. First, high energy electrons are more penetrating than low energy electrons, and thus, they are able to image through thicker samples. Second, the accelerating mode of a radio-frequency linear accelerator provides fast exposures, down to 1 ps, which are ideal for flash radiography, making THEEM well suited to study the evolution of fast material processes under dynamic conditions. Lastly, initial investigations with static objects and during material processing have been performedmore » to investigate the capabilities of this technique.« less

Super-resolution differential interference contrast microscopy by structured illumination.

PubMed

Chen, Jianling; Xu, Yan; Lv, Xiaohua; Lai, Xiaomin; Zeng, Shaoqun

2013-01-14

We propose a structured illumination differential interference contrast (SI-DIC) microscopy, breaking the diffraction resolution limit of differential interference contrast (DIC) microscopy. SI-DIC extends the bandwidth of coherent transfer function of the DIC imaging system, thus the resolution is improved. With 0.8 numerical aperture condenser and objective, the reconstructed SI-DIC image of 53 nm polystyrene beads reveals lateral resolution of approximately 190 nm, doubling that of the conventional DIC image. We also demonstrate biological observations of label-free cells with improved spatial resolution. The SI-DIC microscopy can provide sub-diffraction resolution and high contrast images with marker-free specimens, and has the potential for achieving sub-diffraction resolution quantitative phase imaging.

Fractal and Fourier analysis of the hepatic sinusoidal network in normal and cirrhotic rat liver

PubMed Central

Gaudio, Eugenio; Chaberek, Slawomir; Montella, Andrea; Pannarale, Luigi; Morini, Sergio; Novelli, Gilnardo; Borghese, Federica; Conte, Davide; Ostrowski, Kazimierz

2005-01-01

The organization of the hepatic microvascular network has been widely studied in recent years, especially with regard to cirrhosis. This research has enabled us to recognize the distinctive vascular patterns in the cirrhotic liver, compared with the normal liver, which may explain the cause of liver dysfunction and failure. The aim of this study was to compare normal and cirrhotic rat livers by means of a quantitative mathematical approach based on fractal and Fourier analyses performed on photomicrographs and therefore on discriminant analysis. Vascular corrosion casts of livers belonging to the following three experimental groups were studied by scanning electron microscopy: normal rats, CCl4-induced cirrhotic rats and cirrhotic rats after ligation of the bile duct. Photomicrographs were taken at a standard magnification; these images were used for the mathematical analysis. Our experimental design found that use of these different analyses reaches an efficiency of over 94%. Our analyses demonstrated a higher complexity of the normal hepatic sinusoidal network in comparison with the cirrhotic network. In particular, the morphological changes were more marked in the animals with bile duct-ligation cirrhosis compared with animals with CCl4-induced cirrhosis. The present findings based on fractal and Fourier analysis could increase our understanding of the pathophysiological alterations of the liver, and may have a diagnostic value in future clinical research. PMID:16050897

Analysis of liquid suspensions using scanning electron microscopy in transmission: estimation of the water film thickness using Monte-Carlo simulations.

PubMed

Xiao, J; Foray, G; Masenelli-Varlot, K

2018-02-01

Environmental scanning electron microscopy (ESEM) allows the observation of liquids under specific conditions of pressure and temperature. Moreover, when working in the transmission mode, that is in scanning transmission electron microscopy (STEM), nano-objects can be analysed inside a liquid. The contrast in the images is mass-thickness dependent as in STEM-in-TEM (transmission electron microscopy) using closed cells. However, in STEM-in-ESEM, as the liquid-vapour equilibrium is kept dynamically, the thickness of the water droplet remains unknown. In this paper, the contrasts measured in the experimental images are compared with calculations using Monte-Carlo simulations in order to estimate the thickness of water. Two examples are given. On gold nanoparticles, the thickness of a thick film can be estimated thanks to a contrast inversion. On core-shell latex particles, the grey level of the shell compared with those of the core and of the water film gives a relatively precise measurement of the water film thickness. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Anaerobic choline metabolism in microcompartments promotes growth and swarming of Proteus mirabilis.

PubMed

Jameson, Eleanor; Fu, Tiantian; Brown, Ian R; Paszkiewicz, Konrad; Purdy, Kevin J; Frank, Stefanie; Chen, Yin

2016-09-01

Gammaproteobacteria are important gut microbes but only persist at low levels in the healthy gut. The ecology of Gammaproteobacteria in the gut environment is poorly understood. Here, we demonstrate that choline is an important growth substrate for representatives of Gammaproteobacteria. Using Proteus mirabilis as a model, we investigate the role of choline metabolism and demonstrate that the cutC gene, encoding a choline-trimethylamine lyase, is essential for choline degradation to trimethylamine by targeted mutagenesis of cutC and subsequent complementation experiments. Proteus mirabilis can rapidly utilize choline to enhance growth rate and cell yield in broth culture. Importantly, choline also enhances swarming-associated colony expansion of P. mirabilis under anaerobic conditions on a solid surface. Comparative transcriptomics demonstrated that choline not only induces choline-trimethylamine lyase but also genes encoding shell proteins for the formation of bacterial microcompartments. Subsequent analyses by transmission electron microscopy confirmed the presence of such novel microcompartments in cells cultivated in liquid broth and hyper-flagellated swarmer cells from solid medium. Together, our study reveals choline metabolism as an adaptation strategy for P. mirabilis and contributes to better understand the ecology of this bacterium in health and disease. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Efficacy of terbinafine compared to lanoconazole and luliconazole in the topical treatment of dermatophytosis in a guinea pig model.

PubMed

Ghannoum, M A; Long, L; Kim, H G; Cirino, A J; Miller, A R; Mallefet, P

2010-05-01

The in vivo efficacy of terbinafine was compared to lanoconazole and luliconazole in the topical treatment of dermatophytosis caused by Trichophyton mentagrophytes using a guinea pig model. Topical antifungal treatment commenced three days post-infection, and each agent was applied once daily for seven consecutive days. Upon completion of the treatment period, evaluations of clinical and mycological efficacies were performed, as was scanning electron microscopy (SEM) analyses. Data showed that while all tested antifungals demonstrated significant mycological efficacy in terms of eradicating the fungi over untreated control, terbinafine and luliconazole showed superior clinical efficacy compared to lanoconazole (P-values < 0.001 & 0.003, respectively). Terbinafine demonstrated the highest clinical percent efficacy. SEM analysis revealed hairs from terbinafine and lanoconazole-treated animals had near complete clearance of fungi, while samples from luliconazole-treated animals were covered with debris and few conidia. This study demonstrates that, in general, terbinafine possessed similar efficacy to lanoconazole and luliconazole in the treatment of dermatophytosis. Terbinafine tended to have superior clinical efficacy compared to the azoles tested, although this difference was not statistically significant against luliconazole. This apparent superiority may be due to the fungicidal activity of terbinafine compared to the fungistatic effect of the other two drugs.

Ultrastructural analysis of cell component distribution in the apical cell of Ceratodon protonemata

NASA Technical Reports Server (NTRS)

Walker, L. M.; Sack, F. D.

1995-01-01

A distinctive feature of tip-growing plant cells is that cell components are distributed differentially along the length of the cell, although most ultrastructural analyses have been qualitative. The longtitudinal distribution of cell components was studied both qualitatively and quantitatively in the apical cell of dark-grown protonemata of the moss Ceratodon. The first 35 micrometers of the apical cell was analyzed stereologically using transmission electron microscopy. There were four types of distributions along the cell's axis, three of them differential: (1) tubular endoplasmic reticulum was evenly distributed, (2) cisternal endoplasmic reticulum and Golgi vesicles were distributed in a tip-to-base gradient, (3) plastids, vacuoles, and Golgi stacks were enriched in specific areas, although the locations of the enrichments varied, and (4) mitochondria were excluded in the tip-most 5 micrometers and evenly distributed throughout the remaining 30 micrometers. This study provides one of the most comprehensive quantitative, ultrastructural analyses of the distribution of cell components in the apex of any tip-growing plant cell. The finding that almost every component had its own spatial arrangement demonstrates the complexity of the organization and regulation of the distribution of components in tip-growing cells.

Analysis of variability in additive manufactured open cell porous structures.

PubMed

Evans, Sam; Jones, Eric; Fox, Pete; Sutcliffe, Chris

2017-06-01

In this article, a novel method of analysing build consistency of additively manufactured open cell porous structures is presented. Conventionally, methods such as micro computed tomography or scanning electron microscopy imaging have been applied to the measurement of geometric properties of porous material; however, high costs and low speeds make them unsuitable for analysing high volumes of components. Recent advances in the image-based analysis of open cell structures have opened up the possibility of qualifying variation in manufacturing of porous material. Here, a photogrammetric method of measurement, employing image analysis to extract values for geometric properties, is used to investigate the variation between identically designed porous samples measuring changes in material thickness and pore size, both intra- and inter-build. Following the measurement of 125 samples, intra-build material thickness showed variation of ±12%, and pore size ±4% of the mean measured values across five builds. Inter-build material thickness and pore size showed mean ranges higher than those of intra-build, ±16% and ±6% of the mean material thickness and pore size, respectively. Acquired measurements created baseline variation values and demonstrated techniques suitable for tracking build deviation and inspecting additively manufactured porous structures to indicate unwanted process fluctuations.

Multimodality optical imaging of embryonic heart microstructure

PubMed Central

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H.; Boudoux, Caroline; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

2009-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 µm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass. PMID:18163837

Multimodality optical imaging of embryonic heart microstructure.

PubMed

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H; Boudoux, Caroline; Vakoc, Benjamin J; Bouma, Brett E; Tearney, Guillermo J

2007-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

PubMed Central

Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

2010-01-01

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

Laboratory Exercise for Studying the Morphology of Heat-Denatured and Amyloid Aggregates of Lysozyme by Atomic Force Microscopy

ERIC Educational Resources Information Center

Gokalp, Sumeyra; Horton, William; Jónsdóttir-Lewis, Elfa B.; Foster, Michelle; Török, Marianna

2018-01-01

To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost…

Evaluation of a hybrid pixel detector for electron microscopy.

PubMed

Faruqi, A R; Cattermole, D M; Henderson, R; Mikulec, B; Raeburn, C

2003-04-01

We describe the application of a silicon hybrid pixel detector, containing 64 by 64 pixels, each 170 microm(2), in electron microscopy. The device offers improved resolution compared to CCDs along with faster and noiseless readout. Evaluation of the detector, carried out on a 120 kV electron microscope, demonstrates the potential of the device.

Fourier ptychographic microscopy at telecommunication wavelengths using a femtosecond laser

NASA Astrophysics Data System (ADS)

Ahmed, Ishtiaque; Alotaibi, Maged; Skinner-Ramos, Sueli; Dominguez, Daniel; Bernussi, Ayrton A.; de Peralta, Luis Grave

2017-12-01

We report the implementation of the Fourier Ptychographic Microscopy (FPM) technique, a phase retrieval technique, at telecommunication wavelengths using a low-coherence ultrafast pulsed laser source. High quality images, near speckle-free, were obtained with the proposed approach. We demonstrate that FPM can also be used to image periodic features through a silicon wafer.

Chemical Silver Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

NASA Technical Reports Server (NTRS)

Vikram, Chandra S.; Witherow, William K.

1998-01-01

We report what is believed to be the first experimental demonstration of silver coating by a wet chemical process on tapered fiber tips used in near-field scanning optical microscopy. The process is at room temperature and pressure and takes only a few minutes to complete. Many tips can be simultaneously coated.

Photoacoustic microscopy of single cells employing an intensity-modulated diode laser

NASA Astrophysics Data System (ADS)

Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Dasa, Manoj Kumar; Klar, Thomas A.; Berer, Thomas

2018-02-01

In this work, we employ frequency-domain photoacoustic microscopy to obtain photoacoustic images of labeled and unlabeled cells. The photoacoustic microscope is based on an intensity-modulated diode laser in combination with a focused piezo-composite transducer and allows imaging of labeled cells without severe photo-bleaching. We demonstrate that frequency-domain photoacoustic microscopy realized with a diode laser is capable of recording photoacoustic images of single cells with sub-µm resolution. As examples, we present images of undyed human red blood cells, stained human epithelial cells, and stained yeast cells.

Boundary fitting based segmentation of fluorescence microscopy images

NASA Astrophysics Data System (ADS)

Lee, Soonam; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2015-03-01

Segmentation is a fundamental step in quantifying characteristics, such as volume, shape, and orientation of cells and/or tissue. However, quantification of these characteristics still poses a challenge due to the unique properties of microscopy volumes. This paper proposes a 2D segmentation method that utilizes a combination of adaptive and global thresholding, potentials, z direction refinement, branch pruning, end point matching, and boundary fitting methods to delineate tubular objects in microscopy volumes. Experimental results demonstrate that the proposed method achieves better performance than an active contours based scheme.

Application of laser scanning speckle-microscopy for high-resolution express diagnostics of chlamydial infection

NASA Astrophysics Data System (ADS)

Ulyanov, Sergey; Larionova, Olga; Ulianova, Onega; Zaitsev, Sergey; Saltykov, Yury; Polyanina, Tatiana; Lyapina, Anna; Filonova, Nadezhda; Subbotina, Irina; Kalduzova, Irina; Utz, Sergey; Moiseeva, Yulia; Feodorova, Valentina

2018-04-01

Method of speckle-microscopy has been adapted to the problem of detection of Chlamydia trachomatis microbial cells in clinical samples. Prototype of laser scanning speckle-microscope has been designed. Spatial resolution and output characteristics of this microscope have been analyzed for the case of scanning of C. trachomatis bacteria inclusions - Elementary Bodies (EBs) inside the human cells, fixed on the glass. It has been demonstrated, that presence of C. trachomatis microbial cells in the sample can be easily detected using speckle microscopy.

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

PubMed Central

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; Shapiro, David; Kirz, Janos; Marchesini, Stefano; Neiman, Aaron M.; Turner, Joshua J.; Jacobsen, Chris

2010-01-01

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11–13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy. PMID:20368463

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

DOE PAGES

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; ...

2010-04-20

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane andmore » freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.« less

Rotary-scanning optical resolution photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Qi, Weizhi; Xi, Lei

2016-10-01

Optical resolution photoacoustic microscopy (ORPAM) is currently one of the fastest evolving photoacoustic imaging modalities. It has a comparable spatial resolution to pure optical microscopic techniques such as epifluorescence microscopy, confocal microscopy, and two-photon microscopy, but also owns a deeper penetration depth. In this paper, we report a rotary-scanning (RS)-ORPAM that utilizes a galvanometer scanner integrated with objective to achieve rotary laser scanning. A 15 MHz cylindrically focused ultrasonic transducer is mounted onto a motorized rotation stage to follow optical scanning traces synchronously. To minimize the loss of signal to noise ratio, the acoustic focus is precisely adjusted to reach confocal with optical focus. Black tapes and carbon fibers are firstly imaged to evaluate the performance of the system, and then in vivo imaging of vasculature networks inside the ears and brains of mice is demonstrated using this system.

Solid-phase data from cores at the proposed Dewey Burdock uranium in-situ recovery mine, near Edgemont, South Dakota

USGS Publications Warehouse

Johnson, Raymond H.; Diehl, Sharon F.; Benzel, William M.

2013-01-01

This report releases solid-phase data from cores at the proposed Dewey Burdock uranium in-situ recovery site near Edgemont, South Dakota. These cores were collected by Powertech Uranium Corporation, and material not used for their analyses were given to the U.S. Geological Survey for additional sampling and analyses. These additional analyses included total carbon and sulfur, whole rock acid digestion for major and trace elements, 234U/238U activity ratios, X-ray diffraction, thin sections, scanning electron microscopy analyses, and cathodoluminescence. This report provides the methods and data results from these analyses along with a short summary of observations.

Anomalous Diffusion of Single Particles in Cytoplasm

PubMed Central

Regner, Benjamin M.; Vučinić, Dejan; Domnisoru, Cristina; Bartol, Thomas M.; Hetzer, Martin W.; Tartakovsky, Daniel M.; Sejnowski, Terrence J.

2013-01-01

The crowded intracellular environment poses a formidable challenge to experimental and theoretical analyses of intracellular transport mechanisms. Our measurements of single-particle trajectories in cytoplasm and their random-walk interpretations elucidate two of these mechanisms: molecular diffusion in crowded environments and cytoskeletal transport along microtubules. We employed acousto-optic deflector microscopy to map out the three-dimensional trajectories of microspheres migrating in the cytosolic fraction of a cellular extract. Classical Brownian motion (BM), continuous time random walk, and fractional BM were alternatively used to represent these trajectories. The comparison of the experimental and numerical data demonstrates that cytoskeletal transport along microtubules and diffusion in the cytosolic fraction exhibit anomalous (nonFickian) behavior and posses statistically distinct signatures. Among the three random-walk models used, continuous time random walk provides the best representation of diffusion, whereas microtubular transport is accurately modeled with fractional BM. PMID:23601312

Analysis system of submicron particle tracks in the fine-grained nuclear emulsion by a combination of hard x-ray and optical microscopy

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

Naka, T., E-mail: naka@flab.phys.nagoya-u.ac.jp; Institute for Advanced Research, Nagoya University, Aichi 464-8602; Asada, T.

Analyses of nuclear emulsion detectors that can detect and identify charged particles or radiation as tracks have typically utilized optical microscope systems because the targets have lengths from several μm to more than 1000 μm. For recent new nuclear emulsion detectors that can detect tracks of submicron length or less, the current readout systems are insufficient due to their poor resolution. In this study, we developed a new system and method using an optical microscope system for rough candidate selection and the hard X-ray microscope system at SPring-8 for high-precision analysis with a resolution of better than 70 nm resolution.more » Furthermore, we demonstrated the analysis of submicron-length tracks with a matching efficiency of more than 99% and position accuracy of better than 5 μm. This system is now running semi-automatically.« less

Real-time observation of slipping and rolling events in DLC wear nanoparticles.

PubMed

Sato, Takaaki; Nabeya, Shinsuke; Menon, Vivek; Ishida, Tadashi; Kometani, Reo; Fujita, Hiroyuki

2018-08-10

Real-time observation of the actual contact area between surface interfaces at the nanoscale enables more precise examination of what happens during friction. We have combined micro electro mechanical system actuators and transmission electron microscopy (TEM) observation, to both apply and measure forces across nanoscale junctions and contacts. This custom-designed experimental system can measure the true surface area of a contact site from a lateral viewpoint, while simultaneously measuring the friction force. We scratched surfaces coated with diamond like carbon, a classical solid lubricant, and observed the formation of wear particles that slipped and rolled between the interface. TEM images showed that the shape of the surface at the nanoscale underwent permanent deformation when acted upon with forces as low as several tens of nano newtons. The results demonstrated the limitations of friction analyses relying on friction force measurements without real-time surface profiling.

Quantitative microscopy of the lung: a problem-based approach. Part 2: stereological parameters and study designs in various diseases of the respiratory tract.

PubMed

Mühlfeld, Christian; Ochs, Matthias

2013-08-01

Design-based stereology provides efficient methods to obtain valuable quantitative information of the respiratory tract in various diseases. However, the choice of the most relevant parameters in a specific disease setting has to be deduced from the present pathobiological knowledge. Often it is difficult to express the pathological alterations by interpretable parameters in terms of volume, surface area, length, or number. In the second part of this companion review article, we analyze the present pathophysiological knowledge about acute lung injury, diffuse parenchymal lung diseases, emphysema, pulmonary hypertension, and asthma to come up with recommendations for the disease-specific application of stereological principles for obtaining relevant parameters. Worked examples with illustrative images are used to demonstrate the work flow, estimation procedure, and calculation and to facilitate the practical performance of equivalent analyses.

[Biliary calculi resistant to dissolution with bile acids: their heterogeneous composition and diversity of treatment response].

PubMed

Ruíz de Aguiar, A; Medina, J A; Garrido, G; Villacorta, J; Berenguer, J

1992-05-01

We have studied thirteen biliary stones resistant to biliary acids, using technical methods of stereomicroscopy, scanning electronic microscopy and EDX analyses. We have investigated changes on surface. Three biliary stones did not change and were considered resistant. Seven biliary stones appear partially dissolved and we observed many irregularities on surface and/or concentric dips in relation with cholesterol dissolution. In six cases, biliary pigment alternates with cholesterol. In three cases we observed a calcium carbonate coat on surface. One case included organic fibers. One biliary stone showed cholesterol with spherical bodies of calcium carbonate and pigment. It was a relapsed case of combined treatment. Three stones are composed of small black portions of polymerized calcium bilirubinate, rich in copper and iron. Our results demonstrate that biliary stones previously selected for treatment are a heterogeneous group. Because of this fact we get variable and unpredictable results.

An evaluation of potential occupational exposure to asbestiform amphiboles near a former vermiculite mine.

PubMed

Hart, Julie F; Spear, Terry M; Ward, Tony J; Baldwin, Caitlan E; Salo, Marissa N; Elashheb, Mohamed I

2009-01-01

Amphibole asbestos (AA) has been detected on the surface of tree bark in forests neighboring an abandoned vermiculite mine near Libby, Montana. In the present study, simulations were performed to assess potential AA exposure associated with United States Department of Agriculture Forest Service (FS) occupational activities. Bark samples were collected prior, and personal breathing zone (PBZ) and Tyvek clothing wipe samples were collected during and immediately after trials that simulated FS activities. Transmission electron microscopy (TEM) analyses revealed AA bark concentrations up to 15 million structures per square centimeter (s/cm(2)). AA was detected in 25% of the PBZ TEM samples. AA was detected on wipe samples collected from all activities evaluated. This research demonstrates the potential for airborne exposure and transport of AA in the Kootenai National Forest. These findings are especially relevant to those that work in the area and to the general public who may conduct recreational activities.

Novel near-infrared emission from crystal defects in MoS2 multilayer flakes.

PubMed

Fabbri, F; Rotunno, E; Cinquanta, E; Campi, D; Bonnini, E; Kaplan, D; Lazzarini, L; Bernasconi, M; Ferrari, C; Longo, M; Nicotra, G; Molle, A; Swaminathan, V; Salviati, G

2016-10-04

The structural defects in two-dimensional transition metal dichalcogenides, including point defects, dislocations and grain boundaries, are scarcely considered regarding their potential to manipulate the electrical and optical properties of this class of materials, notwithstanding the significant advances already made. Indeed, impurities and vacancies may influence the exciton population, create disorder-induced localization, as well as modify the electrical behaviour of the material. Here we report on the experimental evidence, confirmed by ab initio calculations, that sulfur vacancies give rise to a novel near-infrared emission peak around 0.75 eV in exfoliated MoS 2 flakes. In addition, we demonstrate an excess of sulfur vacancies at the flake's edges by means of cathodoluminescence mapping, aberration-corrected transmission electron microscopy imaging and electron energy loss analyses. Moreover, we show that ripplocations, extended line defects peculiar to this material, broaden and redshift the MoS 2 indirect bandgap emission.

Chromatin organization regulates viral egress dynamics

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

Aho, Vesa; Myllys, Markko; Ruokolainen, Visa

Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walkmore » modelling of herpes simplex virus 1–sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.« less

Trypanosoma cruzi: activity of essential oils from Achillea millefolium L., Syzygium aromaticum L. and Ocimum basilicum L. on epimastigotes and trypomastigotes.

PubMed

Santoro, Giani F; Cardoso, Maria G; Guimarães, Luiz Gustavo L; Mendonça, Lidiany Z; Soares, Maurilio J

2007-07-01

Trypanocidal activity of clove (Syzygium aromaticum L.), basil (Ocimum basilicum L.) and yarrow (Achillea millefolium L.) essential oils and some of their constituents (eugenol and linalool) was investigated on Trypanosoma cruzi epimastigote and bloodstream trypomastigote forms. Steam distillation was used to isolate the essential oils, with chemical analyses performed by gas chromatography (GC) and GC coupled to mass spectrometry (GC-MS). The IC(50) (concentration that inhibits 50% parasite growth) of the oils and constituents upon T. cruzi was determined by cell counting in a Neubauer chamber. Cell morphology alterations were observed by scanning and transmission electron microscopy. Treatment with oils and constituents demonstrated that they inhibit parasite growth, with clove essential being the most effective one (IC(50)=99.5 microg/ml for epimastigotes and 57.5 microg/ml for trypomastigotes). Ultrastructural alterations were observed mainly in the nucleus.

Novel near-infrared emission from crystal defects in MoS2 multilayer flakes

PubMed Central

Fabbri, F.; Rotunno, E.; Cinquanta, E.; Campi, D.; Bonnini, E.; Kaplan, D.; Lazzarini, L.; Bernasconi, M.; Ferrari, C.; Longo, M.; Nicotra, G.; Molle, A.; Swaminathan, V.; Salviati, G.

2016-01-01

The structural defects in two-dimensional transition metal dichalcogenides, including point defects, dislocations and grain boundaries, are scarcely considered regarding their potential to manipulate the electrical and optical properties of this class of materials, notwithstanding the significant advances already made. Indeed, impurities and vacancies may influence the exciton population, create disorder-induced localization, as well as modify the electrical behaviour of the material. Here we report on the experimental evidence, confirmed by ab initio calculations, that sulfur vacancies give rise to a novel near-infrared emission peak around 0.75 eV in exfoliated MoS2 flakes. In addition, we demonstrate an excess of sulfur vacancies at the flake's edges by means of cathodoluminescence mapping, aberration-corrected transmission electron microscopy imaging and electron energy loss analyses. Moreover, we show that ripplocations, extended line defects peculiar to this material, broaden and redshift the MoS2 indirect bandgap emission. PMID:27698425

Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins

PubMed Central

Ichikawa, Muneyoshi; Liu, Dinan; Kastritis, Panagiotis L.; Basu, Kaustuv; Hsu, Tzu Chin; Yang, Shunkai; Bui, Khanh Huy

2017-01-01

Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule. PMID:28462916

Tunable Single-Cell Extraction for Molecular Analyses.

PubMed

Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

2016-07-14

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. Copyright © 2016 Elsevier Inc. All rights reserved.

Bio-inspired, colorful, flexible, defrostable light-scattering hybrid films for the effective distribution of LED light.

PubMed

An, Seongpil; Jo, Hong Seok; Kim, Yong Il; Song, Kyo Yong; Kim, Min-Woo; Lee, Kyu Bum; Yarin, Alexander L; Yoon, Sam S

2017-07-06

Bioluminescent jellyfish has a unique structure derived from fiber/polymer interfaces that is advantageous for effective light scattering in the dark, deep sea water. Herein, we demonstrate the fabrication of bio-inspired hybrid films by mimicry of the jellyfish's structure, leading to excellent light-scattering performance and defrosting capability. A haze value reaching 59.3% and a heating temperature of up to 292 °C were achieved with the films. Accordingly, the developed surface constitutes an attractive optical device for lighting applications, especially for street or vehicle luminaries for freezing Arctic-climate countries. The morphological details of the hybrid films were revealed by scanning electron microscopy. The light-scattering properties of these films were examined by ultraviolet-visible-infrared spectrophotometry and anti-glare effect analyses. The defrosting performance of the hybrid films was evaluated via heating tests and infra-red observations.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Phase stability and microstructures of high entropy alloys ion irradiated to high doses

NASA Astrophysics Data System (ADS)

Xia, Songqin; Gao, Michael C.; Yang, Tengfei; Liaw, Peter K.; Zhang, Yong

2016-11-01

The microstructures of AlxCoCrFeNi (x = 0.1, 0.75 and 1.5 in molar ratio) high entropy alloys (HEAs) irradiated at room temperature with 3 MeV Au ions at the highest fluence of 105, 91, and 81 displacement per atom, respectively, were studied. Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) analyses show that the initial microstructures and phase composition of all three alloys are retained after ion irradiation and no phase decomposition is observed. Furthermore, it is demonstrated that the disordered face-centered cubic (FCC) and disordered body-centered cubic (BCC) phases show much less defect cluster formation and structural damage than the NiAl-type ordered B2 phase. This effect is explained by higher entropy of mixing, higher defect formation/migration energies, substantially lower thermal conductivity, and higher atomic level stress in the disordered phases.

Chromatin organization regulates viral egress dynamics

DOE PAGES

Aho, Vesa; Myllys, Markko; Ruokolainen, Visa; ...

2017-06-16

Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walkmore » modelling of herpes simplex virus 1–sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.« less

Identification of Peptides in Flowers of Sambucus nigra with Antimicrobial Activity against Aquaculture Pathogens.

PubMed

Álvarez, Claudio Andrés; Barriga, Andrés; Albericio, Fernando; Romero, María Soledad; Guzmán, Fanny

2018-04-27

The elder ( Sambucus spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of Sambucus nigra L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Novel near-infrared emission from crystal defects in MoS2 multilayer flakes

NASA Astrophysics Data System (ADS)

Fabbri, F.; Rotunno, E.; Cinquanta, E.; Campi, D.; Bonnini, E.; Kaplan, D.; Lazzarini, L.; Bernasconi, M.; Ferrari, C.; Longo, M.; Nicotra, G.; Molle, A.; Swaminathan, V.; Salviati, G.

2016-10-01

The structural defects in two-dimensional transition metal dichalcogenides, including point defects, dislocations and grain boundaries, are scarcely considered regarding their potential to manipulate the electrical and optical properties of this class of materials, notwithstanding the significant advances already made. Indeed, impurities and vacancies may influence the exciton population, create disorder-induced localization, as well as modify the electrical behaviour of the material. Here we report on the experimental evidence, confirmed by ab initio calculations, that sulfur vacancies give rise to a novel near-infrared emission peak around 0.75 eV in exfoliated MoS2 flakes. In addition, we demonstrate an excess of sulfur vacancies at the flake's edges by means of cathodoluminescence mapping, aberration-corrected transmission electron microscopy imaging and electron energy loss analyses. Moreover, we show that ripplocations, extended line defects peculiar to this material, broaden and redshift the MoS2 indirect bandgap emission.

A story told by a single nanoparticle in the body fluid: demonstration of dissolution-reprecipitation of nanocrystals in a biological system.

PubMed

Wu, Cheng-Yeu; Young, David; Martel, Jan; Young, John D

2015-01-01

Analysis of the chemical composition of mineral particles found in the body is critical to understand the formation and effects of these entities in vivo. Yet, the possibility that biological fluids may modulate particle composition over time has not been examined. Materials & methods: Mineralo-organic nanoparticles similar to the ones that spontaneously form in human tissues were analyzed using electron microscopy, spectroscopy and proteomic analyses.   We show that the mineralo-organic nanoparticles assimilate various ions and minerals during incubation in ionic solutions simulating body fluids. The particles undergo dissolution-reprecipitation reactions that affect the final protein composition of the particles. The reactions occurring at the mineral-water interface therefore modulate the ionic and organic composition of mineral nanoparticles formed in biological fluids, producing changes that may alter the effects of mineral particles and stones in vivo.

Preparation and characterization of 'green' hybrid clay-dye nanopigments

NASA Astrophysics Data System (ADS)

Kaya, Mehmet; Onganer, Yavuz; Tabak, Ahmet

2015-03-01

We obtained a low cost and abundant nanopigment material composed of Rhodamine B (Rh-B) organic dye compound and Unye bentonite (UB) clay from Turkey. The characterization of the nanopigment was investigated using scanning electron microscopy (SEM), particle size distribution, powder X-ray diffraction (PXRD), Fourier transformed infra-red spectroscopy (FT-IR) and thermal analysis techniques. According to the result of texture analyses, we showed that the particle size distribution (d: 0.5-mean distribution) of Rh-B/UB nanopigment material was around 100 nm diameter. It was also demonstrated that the samples had a particle size around nm diameter in SEM images. As seen in the PXRD and thermal analysis, there is a difference in basal spacing by 1.46° (2θ) and a higher mass loss by 7.80% in the temperature range 200-500 °C compared to the raw bentonite.

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

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

Balke, Nina; Jesse, Stephen; Yu, Pu

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue.

PubMed

Yoshitake, Tadayuki; Giacomelli, Michael G; Cahill, Lucas C; Schmolze, Daniel B; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Fujimoto, James G

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

PubMed Central

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-01-01

Abstract. Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue. PMID:28032121

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

DOE PAGES

Balke, Nina; Jesse, Stephen; Yu, Pu; ...

2016-09-15

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

NASA Astrophysics Data System (ADS)

Yoshitake, Tadayuki; Giacomelli, Michael G.; Cahill, Lucas C.; Schmolze, Daniel B.; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Connolly, James L.; Fujimoto, James G.

2016-12-01

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue.

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

NASA Astrophysics Data System (ADS)

Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

2016-12-01

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

PubMed

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

PubMed Central

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

Boundary segmentation for fluorescence microscopy using steerable filters

NASA Astrophysics Data System (ADS)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Analysing magnetism using scanning SQUID microscopy.

PubMed

Reith, P; Renshaw Wang, X; Hilgenkamp, H

2017-12-01

Scanning superconducting quantum interference device microscopy (SSM) is a scanning probe technique that images local magnetic flux, which allows for mapping of magnetic fields with high field and spatial accuracy. Many studies involving SSM have been published in the last few decades, using SSM to make qualitative statements about magnetism. However, quantitative analysis using SSM has received less attention. In this work, we discuss several aspects of interpreting SSM images and methods to improve quantitative analysis. First, we analyse the spatial resolution and how it depends on several factors. Second, we discuss the analysis of SSM scans and the information obtained from the SSM data. Using simulations, we show how signals evolve as a function of changing scan height, SQUID loop size, magnetization strength, and orientation. We also investigated 2-dimensional autocorrelation analysis to extract information about the size, shape, and symmetry of magnetic features. Finally, we provide an outlook on possible future applications and improvements.

Analysing magnetism using scanning SQUID microscopy

NASA Astrophysics Data System (ADS)

Reith, P.; Renshaw Wang, X.; Hilgenkamp, H.

2017-12-01

Scanning superconducting quantum interference device microscopy (SSM) is a scanning probe technique that images local magnetic flux, which allows for mapping of magnetic fields with high field and spatial accuracy. Many studies involving SSM have been published in the last few decades, using SSM to make qualitative statements about magnetism. However, quantitative analysis using SSM has received less attention. In this work, we discuss several aspects of interpreting SSM images and methods to improve quantitative analysis. First, we analyse the spatial resolution and how it depends on several factors. Second, we discuss the analysis of SSM scans and the information obtained from the SSM data. Using simulations, we show how signals evolve as a function of changing scan height, SQUID loop size, magnetization strength, and orientation. We also investigated 2-dimensional autocorrelation analysis to extract information about the size, shape, and symmetry of magnetic features. Finally, we provide an outlook on possible future applications and improvements.

An in vitro study of the microstructure, composition and nanoindentation mechanical properties of remineralizing human dental enamel

NASA Astrophysics Data System (ADS)

Arsecularatne, J. A.; Hoffman, M.

2014-08-01

This paper describes the results of an in vitro investigation on the interrelations among microstructure, composition and mechanical properties of remineralizing human dental enamel. Polished enamel samples have been demineralized for 10 min in an acetic acid solution (at pH 3) followed by remineralization in human saliva for 30 and 120 min. Microstructure variations of sound, demineralized and remineralized enamel samples have been analysed using focused ion beam, scanning electron microscopy and transmission electron microscopy, while their compositions have been analysed using energy dispersive x-ray. Variations in the mechanical properties of enamel samples have been assessed using nanoindentation. The results reveal that, under the selected conditions, only partial remineralization of the softened enamel surface layer occurs where some pores remain unrepaired. As a result, while the nanoindentation elastic modulus shows an improvement following remineralization, hardness does not.

Morphology of glochidia of Lampsilis higginsi (Bivalvia: Unionidae) compared with three related species

USGS Publications Warehouse

Waller, D.L.; Holland Bartels, L. E.; Mitchell, L.G.

1988-01-01

Glochidia of the endangered unionid mussel Lampsilis higginsi (Lea) are morphologically similar to those of several other species in the upper Mississippi River. Life history details, such as the timing of reproduction and identity of host fish, can be readily studied if the glochidia of L. higginsi can be distinguished from those of related species. Authors used light and scanning electron microscopy and statistical analyses of three shell measurements, shell length, shell height, and hinge length, to compare the glochidia of L. higginsi with those of L. radiata siliquoidea (Barnes), L. ventricosa (Barnes), and Ligumia recta (Lamarck). Glochidia of L. higginsi were differentiated by scanning electron microscopy on the basis of a combined examination of the position of the hinge ligament and the width of dorsal ridges, but were indistinguishable by light microscope examination or by statistical analyses of measurements.

In situ localization of nucleolin in the plant nucleolar matrix.

PubMed

Minguez, A; Moreno Diaz de la Espina, S

1996-01-10

The analysis of isolated nucleolar matrices from onion cells by light and electron microscopy, 2-D separation of proteins, and confocal microscopy has confirmed the existence of an organized nucleolar matrix with a complex protein composition to which are attached the insoluble processing complexes. In the present work, we present evidence from immunoblotting, immunofluorescence, immunogold labeling, and preferential cytochemical staining with bismuth salts that an insoluble fraction of the multifunctional protein nucleolin, is a component of the onion nucleolar matrix, and analyse its ultrastructural distribution in the described domains of the matrix.

Focused Ion Beam Microscopy of ALH84001 Carbonate Disks

NASA Technical Reports Server (NTRS)

Thomas-Keprta, Kathie L.; Clemett, Simon J.; Bazylinski, Dennis A.; Kirschvink, Joseph L.; McKay, David S.; Vali, Hojatollah; Gibson, Everett K., Jr.; Romanek, Christopher S.

2005-01-01

Our aim is to understand the mechanism(s) of formation of carbonate assemblages in ALH84001. A prerequisite is that a detailed characterization of the chemical and physical properties of the carbonate be established. We present here analyses by transmission electron microscopy (TEM) of carbonate thin sections produced by both focused ion beam (FIB) sectioning and ultramicrotomy. Our results suggest that the formation of ALH84001 carbonate assemblages were produced by considerably more complex process(es) than simple aqueous precipitation followed by partial thermal decomposition as proposed by other investigators [e.g., 1-3].

Copper Oxide Precipitates in NBS Standard Reference Material 482

PubMed Central

Windsor, Eric S.; Carlton, Robert A.; Gillen, Greg; Wight, Scott A.; Bright, David S.

2002-01-01

Copper oxide has been detected in the copper containing alloys of NBS Standard Reference Material (SRM) 482. This occurrence is significant because it represents heterogeneity within a standard reference material that was certified to be homogeneous on a micrometer scale. Oxide occurs as elliptically to spherically shaped precipitates whose size differs with alloy composition. The largest precipitates occur in the Au20-Cu80 alloy and range in size from submicrometer up to 2 μm in diameter. Precipitates are observed using light microscopy, electron microscopy, and secondary ion mass spectrometry (SIMS). SIMS has demonstrated that the precipitates are present within all the SRM 482 wires that contain copper. Only the pure gold wire is precipitate free. Initial results from the analysis of the Au20-Cu80 alloy indicate that the percentage of precipitates is less than 1 % by area. Electron probe microanalysis (EPMA) of large (2 μm) precipitates in this same alloy indicates that precipitates are detectable by EPMA and that their composition differs significantly from the certified alloy composition. The small size and low percentage of these oxide precipitates minimizes the impact that they have upon the intended use of this standard for electron probe microanalysis. Heterogeneity caused by these oxide precipitates may however preclude the use of this standard for automated EPMA analyses and other microanalysis techniques. PMID:27446759

Improving Osteoblast Response In Vitro by a Nanostructured Thin Film with Titanium Carbide and Titanium Oxides Clustered around Graphitic Carbon.

PubMed

Longo, Giovanni; Ioannidu, Caterina Alexandra; Scotto d'Abusco, Anna; Superti, Fabiana; Misiano, Carlo; Zanoni, Robertino; Politi, Laura; Mazzola, Luca; Iosi, Francesca; Mura, Francesco; Scandurra, Roberto

2016-01-01

Recently, we introduced a new deposition method, based on Ion Plating Plasma Assisted technology, to coat titanium implants with a thin but hard nanostructured layer composed of titanium carbide and titanium oxides, clustered around graphitic carbon. The nanostructured layer has a double effect: protects the bulk titanium against the harsh conditions of biological tissues and in the same time has a stimulating action on osteoblasts. The aim of this work is to describe the biological effects of this layer on osteoblasts cultured in vitro. We demonstrate that the nanostructured layer causes an overexpression of many early genes correlated to proteins involved in bone turnover and an increase in the number of surface receptors for α3β1 integrin, talin, paxillin. Analyses at single-cell level, by scanning electron microscopy, atomic force microscopy, and single cell force spectroscopy, show how the proliferation, adhesion and spreading of cells cultured on coated titanium samples are higher than on uncoated titanium ones. Finally, the chemistry of the layer induces a better formation of blood clots and a higher number of adhered platelets, compared to the uncoated cases, and these are useful features to improve the speed of implant osseointegration. In summary, the nanostructured TiC film, due to its physical and chemical properties, can be used to protect the implants and to improve their acceptance by the bone.

Facile fabrication of Ag3VO4/attapulgite composites for highly efficient visible light-driven photodegradation towards organic dyes and tetracycline hydrochloride

NASA Astrophysics Data System (ADS)

Luo, Yuting; Luo, Jie; Duan, Guorong; Liu, Xiaoheng

2017-12-01

An efficient one-dimensional attapulgite (ATP)-based photocatalyst, Ag3VO4/ATP nanocomposite, was fabricated by a facile deposition precipitation method with well-dispersed Ag3VO4 nanoparticles anchored on the surface of natural ATP fibers. X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and UV-visible diffused reflectance spectroscopy (UV-vis DRS) were employed to investigate the morphologies, structure, and optical property of the prepared photocatalysts. The photocatalytic experiments indicated that the Ag3VO4/ATP nanocomposites exhibited enhanced visible light-driven photocatalytic activity towards the degradation of rhodamine B (RhB), methyl orange (MO), and tetracycline hydrochloride (TCH), of which the 20 wt% Ag3VO4/ATP sample showed superb photocatalytic performance. As demonstrated by N2 adsorption-desorption, photocurrent measurements, electrochemical impedance spectroscopy (EIS), and photoluminescence (PL) spectra analyses, the improved photocatalytic activity arose from the enlarged surface area, the facilitated charge transfer, and the suppressed recombination of photogenerated charge carriers in Ag3VO4/ATP system. Furthermore, radical scavengers trapping experiments and recycling tests were also conducted. This work gives a new insight into fabrication of highly efficient, stable, and cost-effective visible light-driven photocatalyst for practical application in wastewater treatment and environmental remediation.

Potentiometric Zinc Ion Sensor Based on Honeycomb-Like NiO Nanostructures

PubMed Central

Abbasi, Mazhar Ali; Ibupoto, Zafar Hussain; Hussain, Mushtaque; Khan, Yaqoob; Khan, Azam; Nur, Omer; Willander, Magnus

2012-01-01

In this study honeycomb-like NiO nanostructures were grown on nickel foam by a simple hydrothermal growth method. The NiO nanostructures were characterized by field emission electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) techniques. The characterized NiO nanostructures were uniform, dense and polycrystalline in the crystal phase. In addition to this, the NiO nanostructures were used in the development of a zinc ion sensor electrode by functionalization with the highly selective zinc ion ionophore 12-crown-4. The developed zinc ion sensor electrode has shown a good linear potentiometric response for a wide range of zinc ion concentrations, ranging from 0.001 mM to 100 mM, with sensitivity of 36 mV/decade. The detection limit of the present zinc ion sensor was found to be 0.0005 mM and it also displays a fast response time of less than 10 s. The proposed zinc ion sensor electrode has also shown good reproducibility, repeatability, storage stability and selectivity. The zinc ion sensor based on the functionalized NiO nanostructures was also used as indicator electrode in potentiometric titrations and it has demonstrated an acceptable stoichiometric relationship for the determination of zinc ion in unknown samples. The NiO nanostructures-based zinc ion sensor has potential for analysing zinc ion in various industrial, clinical and other real samples. PMID:23202217

Solid oxide membrane-assisted controllable electrolytic fabrication of metal carbides in molten salt.

PubMed

Zou, Xingli; Zheng, Kai; Lu, Xionggang; Xu, Qian; Zhou, Zhongfu

2016-08-15

Silicon carbide (SiC), titanium carbide (TiC), zirconium carbide (ZrC), and tantalum carbide (TaC) have been electrochemically produced directly from their corresponding stoichiometric metal oxides/carbon (MOx/C) precursors by electrodeoxidation in molten calcium chloride (CaCl2). An assembled yttria stabilized zirconia solid oxide membrane (SOM)-based anode was employed to control the electrodeoxidation process. The SOM-assisted controllable electrochemical process was carried out in molten CaCl2 at 1000 °C with a potential of 3.5 to 4.0 V. The reaction mechanism of the electrochemical production process and the characteristics of these produced metal carbides (MCs) were systematically investigated. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy analyses clearly identify that SiC, TiC, ZrC, and TaC carbides can be facilely fabricated. SiC carbide can be controlled to form a homogeneous nanowire structure, while the morphologies of TiC, ZrC, and TaC carbides exhibit porous nodular structures with micro/nanoscale particles. The complex chemical/electrochemical reaction processes including the compounding, electrodeoxidation, dissolution-electrodeposition, and in situ carbonization processes in molten CaCl2 are also discussed. The present results preliminarily demonstrate that the molten salt-based SOM-assisted electrodeoxidation process has the potential to be used for the facile and controllable electrodeoxidation of MOx/C precursors to micro/nanostructured MCs, which can potentially be used for various applications.

Amphotericin B-conjugated polypeptide hydrogels as a novel innovative strategy for fungal infections

NASA Astrophysics Data System (ADS)

Shu, Chang; Li, Tengfei; Yang, Wen; Li, Duo; Ji, Shunli; Ding, Li

2018-03-01

The present work is focused on the design and development of novel amphotericin B (AmB)-conjugated biocompatible and biodegradable polypeptide hydrogels to improve the antifungal activity. Using three kinds of promoting self-assembly groups (2-naphthalene acetic acid (Nap), naproxen (Npx) and dexamethasone (Dex)) and polypeptide sequence (Phe-Phe-Asp-Lys-Tyr, FFDKY), we successfully synthesized the Nap-FFDK(AmB)Y gels, Npx-FFDK(AmB)Y gels and Dex-FFDK(AmB)Y gels. The AmB-conjugated hydrogelators are highly soluble in different aqueous solutions. The cryo-transmission electron microscopy and scanning electron microscopy micrographs of hydrogels afford nanofibres with a width of 20-50 nm. Powder X-ray diffraction analyses demonstrate that the crystalline structures of the AmB and Dex are changed into amorphous structures after the formation of hydrogels. Circular dichroism spectra of the solution of blank carriers and the corresponding drug deliveries further help elucidate the molecular arrangement in gel phase, indicating the existence of turn features. The in vitro drug releases suggest that the AmB-conjugated hydrogels are suitable as drug-controlled release vehicles for hydrophobic drugs. The antifungal effect of AmB-conjugated hydrogels significantly exhibits the antifungal activity against Candida albicans. The results of the present study indicated that the AmB-conjugated hydrogels are suitable carriers for poorly water soluble drugs and for enhancement of therapeutic efficacy of antifungal drugs.

Effects of sub-minimum inhibitory concentrations of antimicrobial agents on Streptococcus mutans biofilm formation.

PubMed

Dong, Liping; Tong, Zhongchun; Linghu, Dake; Lin, Yuan; Tao, Rui; Liu, Jun; Tian, Yu; Ni, Longxing

2012-05-01

Many studies have demonstrated that sub-minimum inhibitory concentrations (sub-MICs) of antimicrobial agents can inhibit bacterial biofilm formation. However, the mechanisms by which antimicrobial agents at sub-MICs inhibit biofilm formation remain unclear. At present, most studies are focused on Gram-negative bacteria; however, the effects of sub-MICs of antimicrobial agents on Gram-positive bacteria may be more complex. Streptococcus mutans is a major cariogenic bacterium. In this study, the S. mutans growth curve as well as the expression of genes related to S. mutans biofilm formation were evaluated following treatment with 0.5× MIC of chlorhexidine (CHX), tea polyphenols and sodium fluoride (NaF), which are common anticaries agents. The BioFlux system was employed to generate a biofilm under a controlled flow. Morphological changes of the S. mutans biofilm were observed and analysed using field emission scanning electron microscopy and confocal laser scanning microscopy. The results indicated that these three common anticaries agents could significantly upregulate expression of the genes related to S. mutans biofilm formation, and S. mutans exhibited a dense biofilm with an extensive extracellular matrix following treatment with sub-MICs of NaF and CHX. These findings suggest that sub-MICs of anticaries agents favour S. mutans biofilm formation, which might encourage dental caries progression. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Structural Channels and Atomic-Cluster Insertion in CsxBi4Te6 (1 ≤ x ≤ 1.25) As Observed by Aberration-Corrected Scanning Transmission Electron Microscopy.

PubMed

Zhang, Ruixin; Yang, Huaixin; Guo, Cong; Tian, Huanfang; Shi, Honglong; Chen, Genfu; Li, Jianqi

2016-12-19

Microstructural analyses based on aberration-corrected scanning transmission electron microscopy (STEM) observations demonstrate that low-dimensional Cs x Bi 4 Te 6 materials, known to be a novel thermoelectric and superconducting system, contain notable structural channels that go directly along the b axis, which can be partially filled by atom clusters depending on the thermal treatment process. We successfully prepared two series of Cs x Bi 4 Te 6 single-crystalline samples using two different sintering processes. The Cs x Bi 4 Te 6 samples prepared using an air-quenching method show superconductivity at approximately 4 K, while the Cs x Bi 4 Te 6 with the same nominal compositions prepared by slowly cooling are nonsuperconductors. Moreover, atomic structural investigations of typical samples reveal that the structural channels are often empty in superconducting materials; thus, we can represent the superconducting phase as Cs 1-y Bi 4 Te 6 with considering the point defects in the Cs layers. In addition, the channels in the nonsuperconducting crystals are commonly partially occupied by triplet Bi clusters. Moreover, the average structures for these two phases are also different in their monoclinic angles (β), which are estimated to be 102.3° for superconductors and 100.5° for nonsuperconductors.

AFM and SEM study of the effects of etching on IPS-Empress 2 TM dental ceramic

NASA Astrophysics Data System (ADS)

Luo, X.-P.; Silikas, N.; Allaf, M.; Wilson, N. H. F.; Watts, D. C.

2001-10-01

The aim of this study was to investigate the effects of increasing etching time on the surface of the new dental material, IPS-Empress 2 TM glass ceramic. Twenty one IPS-Empress 2 TM glass ceramic samples were made from IPS-Empress 2 TM ingots through lost-wax, hot-pressed ceramic fabrication technology. All samples were highly polished and cleaned ultrasonically for 5 min in acetone before and after etching with 9.6% hydrofluoric acid gel. The etching times were 0, 10, 20, 30, 60, 90 and 120 s respectively. Microstructure was analysed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) was used to evaluate the surface roughness and topography. Observations with SEM showed that etching with hydrofluoric acid resulted in preferential dissolution of glass matrix, and that partially supported crystals within the glass matrix were lost with increasing etching time. AFM measurements indicated that etching increased the surface roughness of the glass-ceramic. A simple least-squares linear regression was used to establish a relationship between surface roughness parameters ( Ra, RMS), and etching time, for which r2>0.94. This study demonstrates the benefits of combining two microscopic methods for a better understanding of the surface. SEM showed the mode of action of hydrofluoric acid on the ceramic and AFM provided valuable data regarding the extent of surface degradation relative to etching time.

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

PubMed

Veys, P; Baeten, V

2010-07-01

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

Analysis of Four Automated Urinalysis Systems Compared to Reference Methods.

PubMed

Bartosova, Kamila; Kubicek, Zdenek; Franekova, Janka; Louzensky, Gustav; Lavrikova, Petra; Jabor, Antonin

2016-11-01

The aim of this study was to compare four automated urinalysis systems: the Iris iQ200 Sprint (Iris Diagnostics, U.S.A.) combined with the Arkray AUTION MAX AX 4030, Iris + AUTION, Arkray AU 4050 (Arkray Global Business, Inc., Japan), Dirui FUS 2000 (Dirui Industrial Co., P.R.C.), and Menarini sediMAX (Menarini, Italy). Urine concentrations of protein and glucose (Iris, Dirui) were compared using reference quantitative analysis on an Abbott Architect c16000. Leukocytes, erythrocytes, epithelia, and casts (Iris, Arkray, Diuri, Menarini) were compared to urine sediment under reference light microscopy, Leica DM2000 (Leica Microsystems GmbH, Germany) with calibrated FastRead plates (Biosigma S.r.l., Italy), using both native and stained preparations. Total protein and glucose levels were measured using the Iris + AUTION system with borderline trueness, while the Dirui analysis revealed worse performances for the protein and glucose measurements. True classifications of leukocytes and erythrocytes were above 85% and 72%, respectively. Kappa statistics revealed a nearly perfect evaluation of leukocytes for all tested systems; the erythrocyte evaluation was nearly perfect for the Iris, Dirui and Arkray analyzers and substantial for the Menarini analyzer. The epithelia identification was connected to high false negativity (above 15%) in the Iris, Arkray, and Menarini analyses. False-negative casts were above 70% for all tested systems. The use of automated urinalysis demonstrated some weaknesses and should be checked by experienced laboratory staff using light microscopy.

Association of the sites of heavy metals with nanoscale carbon in a Kentucky electrostatic precipitator fly ash

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

James C. Hower; Uschi M. Graham; Alan Dozier

2008-11-15

A combination of high-resolution transmission electron microscopy, scanning transmission electron microscopy, and electron energy-loss spectroscopy (HRTEM-STEM-EELS) was used to study fly ashes produced from the combustion of an eastern Kentucky coal at a southeastern-Kentucky wall-fired pulverized coal utility boiler retrofitted for low-NOx combustion. Fly ash was collected from individual hoppers in each row of the electrostatic precipitators (ESP) pollution-control system, with multiple hoppers sampled within each of the three rows. Temperatures within the ESP array range from about 200 {degree}C at the entry to the first row to <150{degree}C at the exit of the third row. HRTEM-STEM-EELS study demonstrated themore » presence of nanoscale (10 s nm) C agglomerates with typical soot-like appearance and others with graphitic fullerene-like nanocarbon structures. The minute carbon agglomerates are typically juxtaposed and intergrown with slightly larger aluminosilicate spheres and often form an ultrathin halo or deposit on the fly ash particles. The STEM-EELS analyses revealed that the nanocarbon agglomerates host even finer (<3 nm) metal and metal oxide particles. Elemental analysis indicated an association of Hg with the nanocarbon. Arsenic, Se, Pb, Co, and traces of Ti and Ba are often associated with Fe-rich particles within the nanocarbon deposits. 57 refs., 5 figs.« less

Characterization of hematite nanoparticles synthesized via two different pathways

NASA Astrophysics Data System (ADS)

Das, Soumya; Hendry, M. Jim

2014-08-01

Hematite is one of the most common and thermodynamically stable iron oxides found in both natural and anthropogenic systems. Owing to its ubiquity, stability, moderate specific surface area, and ability to sequester metals and metalloids from aquatic systems, it has been the subject of a large number of adsorption studies published during the past few decades. Although preparation techniques are known to affect the surface morphology of hematite nanoparticles, the effects of aging under environmentally relevant conditions have yet to be tested with respect to surface morphology, surface area, and adsorptive capacity. We prepared hematite via two different pathways and aged it under highly alkaline conditions encountered in many mill tailings settings. Crystal habits and morphologies of the hematite nanoparticles were analyzed via scanning electron microscopy and transmission electron microscopy. X-ray diffraction, Raman spectroscopy, and Brunauer-Emmett-Teller surface area analyses were also conducted on the hematite nanoparticles before and after aging. The hematite synthesized via an Fe(III) salt solution (average particle size 37 nm) was morphologically and structurally different from the hematite synthesized via ferrihydrite aging (average particle size 144 nm). Overall, our data demonstrate that the crystallinity of hematite produced via ferrihydrite transformation is susceptible to morphological alterations/modifications. In contrast, the hematite formed via hydrolysis of an Fe(III) salt solution remains very stable in terms of structure, size, and morphology even under extreme experimental conditions.

Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus.

PubMed

Marsano, Anna; Wendt, David; Raiteri, Roberto; Gottardi, Riccardo; Stolz, Martin; Wirz, Dieter; Daniels, Alma U; Salter, Donald; Jakob, Marcel; Quinn, Thomas M; Martin, Ivan

2006-12-01

The aim of this study was to demonstrate that differences in the local composition of bi-zonal fibrocartilaginous tissues result in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF-11) and cultured for 4 weeks in mixed flask, a rotary Cell Culture System (RCCS), or statically. Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings, respectively. Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2 microm diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues. In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.

Delivery of vincristine sulfate-conjugated gold nanoparticles using liposomes: a light-responsive nanocarrier with enhanced antitumor efficiency

PubMed Central

Liu, Ying; He, Man; Niu, Mengmeng; Zhao, Yiqing; Zhu, Yuanzhang; Li, Zhenhua; Feng, Nianping

2015-01-01

Rapid drug release at the specific site of action is still a challenge for antitumor therapy. Development of stimuli-responsive hybrid nanocarriers provides a promising strategy to enhance therapeutic effects by combining the unique features of each component. The present study explored the use of drug–gold nanoparticle conjugates incorporated into liposomes to enhance antitumor efficiency. A model drug, vincristine sulfate, was physically conjugated with gold nanoparticles and verified by UV-visible and fourier transform infrared spectroscopy, and differential scanning calorimetry. The conjugates were incorporated into liposomes by film dispersion to yield nanoparticles (113.4 nm) with light-responsive release properties, as shown by in vitro release studies. Intracellular uptake and distribution was studied in HeLa cells using transmission electron microscopy and confocal laser scanning microscopy. This demonstrated liposome internalization and localization in endosomal–lysosomal vesicles. Fluorescence intensity increased in cells exposed to UV light, indicating that this stimulated intracellular drug release; this finding was confirmed by quantitative analyses using flow cytometry. Antitumor efficacy was evaluated in HeLa cells, both in culture and in implants in vivo in nude mice. HeLa cell viability assays showed that light exposure enhanced liposome cytotoxicity and induction of apoptosis. Furthermore, treatment with the prepared liposomes coupled with UV light exposure produced greater antitumor effects in nude mice and reduced side effects, as compared with free vincristine sulfate. PMID:25960649

Pump-probe optical microscopy for imaging nonfluorescent chromophores.

PubMed

Wei, Lu; Min, Wei

2012-06-01

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

In Situ Identification of Nanoparticle Structural Information Using Optical Microscopy.

PubMed

Culver, Kayla S B; Liu, Tingting; Hryn, Alexander J; Fang, Ning; Odom, Teri W

2018-05-11

Diffraction-limited optical microscopy lacks the resolution to characterize directly nanoscale features of single nanoparticles. This paper describes how surprisingly rich structural features of small gold nanostars can be identified using differential interference contrast (DIC) microscopy. First, we established a library of structure-property relationships between nanoparticle shape and DIC optical image and then validated the correlation with electrodynamic simulations and electron microscopy. We found that DIC image patterns of single nanostars could be differentiated between 2D and 3D geometries. Also, DIC images could elucidate the symmetry properties and orientation of nanoparticles. Finally, we demonstrated how this wide-field optical technique can be used for in situ characterization of single nanoparticles rotating at a glass-water interface.

Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM

PubMed Central

Ghorbal, Achraf; Grisotto, Federico; Charlier, Julienne; Palacin, Serge; Goyer, Cédric; Demaille, Christophe; Ben Brahim, Ammar

2013-01-01

This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM) electrode. The capabilities of the AFM-SECM-electrode were tested with the nano-electrografting of vinylic monomers initiated by aryl diazonium salts. Nano-electrochemical and technical processes were thoroughly described, so as to allow experiments reproducing. A plausible explanation of chemical and electrochemical mechanisms, leading to the nano-grafting process, was reported. This combined technique represents the first step towards improved nano-processes for the nano-electrografting. PMID:28348337

Nanoscale Visualization of Elastic Inhomogeneities at TiN Coatings Using Ultrasonic Force Microscopy

NASA Astrophysics Data System (ADS)

Hidalgo, J. A.; Montero-Ocampo, C.; Cuberes, M. T.

2009-12-01

Ultrasonic force microscopy has been applied to the characterization of titanium nitride coatings deposited by physical vapor deposition dc magnetron sputtering on stainless steel substrates. The titanium nitride layers exhibit a rich variety of elastic contrast in the ultrasonic force microscopy images. Nanoscale inhomogeneities in stiffness on the titanium nitride films have been attributed to softer substoichiometric titanium nitride species and/or trapped subsurface gas. The results show that increasing the sputtering power at the Ti cathode increases the elastic homogeneity of the titanium nitride layers on the nanometer scale. Ultrasonic force microscopy elastic mapping on titanium nitride layers demonstrates the capability of the technique to provide information of high value for the engineering of improved coatings.

Solid state protein monolayers: Morphological, conformational, and functional properties

NASA Astrophysics Data System (ADS)

Pompa, P. P.; Biasco, A.; Frascerra, V.; Calabi, F.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; de Waal, E.; Canters, G. W.

2004-12-01

We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

Graphene-enhanced infrared near-field microscopy.

PubMed

Li, Peining; Wang, Tao; Böckmann, Hannes; Taubner, Thomas

2014-08-13

Graphene is a promising two-dimensional platform for widespread nanophotonic applications. Recent theories have predicted that graphene can also enhance evanescent fields for subdiffraction-limited imaging. Here, for the first time we experimentally demonstrate that monolayer graphene offers a 7-fold enhancement of evanescent information, improving conventional infrared near-field microscopy to resolve buried structures at a 500 nm depth with λ/11-resolution.

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

2010-05-01

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

The design and construction of a cost-efficient confocal laser scanning microscope

NASA Astrophysics Data System (ADS)

Xi, Peng; Rajwa, Bartlomiej; Jones, James T.; Robinson, J. Paul

2007-03-01

The optical dissection ability of confocal microscopy makes it a powerful tool for biological materials. However, the cost and complexity of confocal scanning laser microscopy hinders its wide application in education. We describe the construction of a simplified confocal scanning laser microscope and demonstrate three-dimensional projection based on cost-efficient commercial hardware, together with available open source software.

"We Actually Saw Atoms with Our Own Eyes": Conceptions and Convictions in Using the Scanning Tunneling Microscope in Junior High School

ERIC Educational Resources Information Center

Margel, Hannah; Eylon, Bat-Sheva; Scherz, Zahava

2004-01-01

The feasibility and the potential contribution of the scanning tunneling microscopy (STM) in junior high school (JHS) as an instructional tool for learning the particulate nature of matter is described. The use and power of new technologies can probably be demonstrated by the scanning tunneling microscopy (STM).

Flow Microscopy Imaging Is Sensitive to Characteristics of Subvisible Particles in Peginesatide Formulations Associated With Severe Adverse Reactions.

PubMed

Daniels, Austin L; Randolph, Theodore W

2018-05-01

The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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

Timmermans, F. J.; Otto, C.

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

Characterization of human breast cancer by scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

In vivo non-invasive monitoring of collagen remodelling by two-photon microscopy after micro-ablative fractional laser resurfacing.

PubMed

Cicchi, Riccardo; Kapsokalyvas, Dimitrios; Troiano, Michela; Campolmi, Piero; Morini, Cristiano; Massi, Daniela; Cannarozzo, Giovanni; Lotti, Torello; Pavone, Francesco Saverio

2014-11-01

Non-linear optical microscopy is becoming popular as a non-invasive in vivo imaging modality in dermatology. In this study, combined TPF and SHG microscopy were used to monitor collagen remodelling in vivo after micro-ablative fractional laser resurfacing. Papillary dermis of living subjects, covering a wide age range, was imaged immediately before and forty days after treatment. A qualitative visual examination of acquired images demonstrated an age-dependent remodelling effect on collagen. Additional quantitative analysis of new collagen production was performed by means of two image analysis methods. A higher increase in SHG to TPF ratio, corresponding to a stronger treatment effectiveness, was found in older subjects, whereas the effect was found to be negligible in young, and minimal in middle age subjects. Analysis of collagen images also showed a dependence of the treatment effectiveness with age but with controversial results. While the diagnostic potential of in vivo multiphoton microscopy has already been demonstrated for skin cancer and other skin diseases, here we first successfully explore its potential use for a non-invasive follow-up of a laser-based treatment. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Folate-grafted boron nitride nanotubes: possible exploitation in cancer therapy.

PubMed

Ferreira, Tiago H; Marino, Attilio; Rocca, Antonella; Liakos, Ioannis; Nitti, Simone; Athanassiou, Athanassia; Mattoli, Virgilio; Mazzolai, Barbara; de Sousa, Edesia M B; Ciofani, Gianni

2015-03-15

Boron nitride nanotubes (BNNTs) have generated considerable interest among the scientific community because of their unique physical and chemical properties. They present good chemical inertness, high thermal stability, and optimal resistance to oxidation, that make them ideal candidates for biomedical applications, in particular as nanovectors for drug, gene and protein delivery into the cells. In this study, BNNTs were prepared through a synthesis based on a chemical vapor deposition (CVD) method, and thereafter chemically functionalized with folic acid. The obtained nanostructures have been characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The characterization showed efficiently functionalized BNNTs of length of about 1 μm. Furthermore, confocal laser microscopy demonstrated that our nanotubes can be fluorescently-traced under appropriate excitation. Thanks to this property, it has been possible to investigate their internalization by HeLa cells through confocal microscopy, demonstrating that the BNNT up-take clearly increases after the functionalization with folate, a result confirmed by inductively coupled plasma (ICP) assessment of boron content inside the treated cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

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

High resolution computational on-chip imaging of biological samples using sparsity constraint (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rivenson, Yair; Wu, Chris; Wang, Hongda; Zhang, Yibo; Ozcan, Aydogan

2017-03-01

Microscopic imaging of biological samples such as pathology slides is one of the standard diagnostic methods for screening various diseases, including cancer. These biological samples are usually imaged using traditional optical microscopy tools; however, the high cost, bulkiness and limited imaging throughput of traditional microscopes partially restrict their deployment in resource-limited settings. In order to mitigate this, we previously demonstrated a cost-effective and compact lens-less on-chip microscopy platform with a wide field-of-view of >20-30 mm^2. The lens-less microscopy platform has shown its effectiveness for imaging of highly connected biological samples, such as pathology slides of various tissue samples and smears, among others. This computational holographic microscope requires a set of super-resolved holograms acquired at multiple sample-to-sensor distances, which are used as input to an iterative phase recovery algorithm and holographic reconstruction process, yielding high-resolution images of the samples in phase and amplitude channels. Here we demonstrate that in order to reconstruct clinically relevant images with high resolution and image contrast, we require less than 50% of the previously reported nominal number of holograms acquired at different sample-to-sensor distances. This is achieved by incorporating a loose sparsity constraint as part of the iterative holographic object reconstruction. We demonstrate the success of this sparsity-based computational lens-less microscopy platform by imaging pathology slides of breast cancer tissue and Papanicolaou (Pap) smears.

Nanoscale quantification of intracellular element concentration by X-ray fluorescence microscopy combined with X-ray phase contrast nanotomography

NASA Astrophysics Data System (ADS)

Gramaccioni, Chiara; Yang, Yang; Procopio, Alessandra; Pacureanu, Alexandra; Bohic, Sylvain; Malucelli, Emil; Iotti, Stefano; Farruggia, Giovanna; Bukreeva, Inna; Notargiacomo, Andrea; Fratini, Michela; Valenti, Piera; Rosa, Luigi; Berlutti, Francesca; Cloetens, Peter; Lagomarsino, Stefano

2018-01-01

We present here a correlative X-ray microscopy approach for quantitative single cell imaging of molar concentrations. By combining the elemental content provided by X-ray fluorescence microscopy and the morphology information extracted from X-ray phase nanotomography, we determine the intracellular molarity distributions. This correlative method was demonstrated on a freeze-dried human phagocytic cell to obtain the absolute elemental concentration maps of K, P, and Fe. The cell morphology results showed a very good agreement with atomic-force microscopy measurements. This work opens the way for non-destructive single cell chemical analysis down to the sub-cellular level using exclusively synchrotron radiation techniques. It will be of high interest in the case where it is difficult to access the morphology using atomic-force microscopy, for example, on frozen-hydrated cells or tissues.

Intravital hybrid optical-optoacoustic microscopy based on fiber-Bragg interferometry

NASA Astrophysics Data System (ADS)

Shnaiderman, Rami; Wissmeyer, Georg; Seeger, Markus; Estrada, Hector; Ntziachristos, Vasilis

2018-02-01

Optoacoustic microscopy (OAM) has enabled high-resolution, label-free imaging of tissues at depths not achievable with purely optical microscopy. However, widespread implementation of OAM into existing epi-illumination microscopy setups is often constrained by the performance and size of the commonly used piezoelectric ultrasound detectors. In this work, we introduce a novel acoustic detector based on a π-phase-shifted fiber Bragg grating (π-FBG) interferometer embedded inside an ellipsoidal acoustic cavity. The cavity enables seamless integration of epi-illumination OAM into existing microscopy setups by decoupling the acoustic and optical paths between the microscope objective and the sample. The cavity also acts as an acoustic condenser, boosting the sensitivity of the π-FBG and enabling cost effective CW-laser interrogation technique. We characterize the sensor's sensitivity and bandwidth and demonstrate hybrid OAM and second-harmonic imaging of phantoms and mouse tissue in vivo.

Applying Superresolution Localization-Based Microscopy to Neurons

PubMed Central

ZHONG, HAINING

2016-01-01

Proper brain function requires the precise localization of proteins and signaling molecules on a nanometer scale. The examination of molecular organization at this scale has been difficult in part because it is beyond the reach of conventional, diffraction-limited light microscopy. The recently developed method of superresolution, localization-based fluorescent microscopy (LBM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), has demonstrated a resolving power at a 10 nm scale and is poised to become a vital tool in modern neuroscience research. Indeed, LBM has revealed previously unknown cellular architectures and organizational principles in neurons. Here, we discuss the principles of LBM, its current applications in neuroscience, and the challenges that must be met before its full potential is achieved. We also present the unpublished results of our own experiments to establish a sample preparation procedure for applying LBM to study brain tissue. PMID:25648102

Bacterial cell identification in differential interference contrast microscopy images.

PubMed

Obara, Boguslaw; Roberts, Mark A J; Armitage, Judith P; Grau, Vicente

2013-04-23

Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins.

Crystals seen on CSF microscopy in a case of suspected subarachnoid haemorrhage

PubMed Central

Weiand, Daniel; Hanning, Ian; Mouhamadou, Moussa; Wearmouth, Debbie

2015-01-01

Although crystals are rarely identified on cerebrospinal fluid (CSF) microscopy, their presence can be of significant diagnostic value. We report a case of oxalate crystals seen on CSF microscopy of a 43-year-old woman. The patient presented with headaches, nausea and vomiting. CT of the head showed a small focus of hyper-density, suspicious of haemorrhage, in the right side of the pontine cistern. CSF cell count was within the normal range. Although no organisms were seen on microscopy, copious oxalate crystals were seen. The same crystals were seen on microscopy of CSF collected in a fluoride oxalate container used for glucose analysis. A follow-up contrast-enhanced CT angiogram did not demonstrate any abnormalities. It transpired that excess CSF had been collected into a fluoride oxalate container. This had subsequently been decanted into a plain container for microbiological analysis. Correct specimen collection should be emphasised when teaching lumbar puncture technique. PMID:26139652

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

NASA Astrophysics Data System (ADS)

Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

2013-03-01

Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

Probing Membrane Order and Topography in Supported Lipid Bilayers by Combined Polarized Total Internal Reflection Fluorescence-Atomic Force Microscopy

PubMed Central

Oreopoulos, John; Yip, Christopher M.

2009-01-01

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C20 or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions. PMID:19254557

Schottky barrier height measurements of Cu/Si(001), Ag/Si(001), and Au/Si(001) interfaces utilizing ballistic electron emission microscopy and ballistic hole emission microscopy

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

Balsano, Robert; Matsubayashi, Akitomo; LaBella, Vincent P., E-mail: vlabella@albany.edu

2013-11-15

The Schottky barrier heights of both n and p doped Cu/Si(001), Ag/Si(001), and Au/Si(001) diodes were measured using ballistic electron emission microscopy and ballistic hole emission microscopy (BHEM), respectively. Measurements using both forward and reverse ballistic electron emission microscopy (BEEM) and (BHEM) injection conditions were performed. The Schottky barrier heights were found by fitting to a linearization of the power law form of the Bell-Kaiser BEEM model. The sum of the n-type and p-type barrier heights are in good agreement with the band gap of silicon and independent of the metal utilized. The Schottky barrier heights are found to bemore » below the region of best fit for the power law form of the BK model, demonstrating its region of validity.« less

Scanning ultrafast electron microscopy.

PubMed

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

2010-08-24

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

External cervical resorption: an analysis using cone beam and microfocus computed tomography and scanning electron microscopy.

PubMed

Gunst, V; Mavridou, A; Huybrechts, B; Van Gorp, G; Bergmans, L; Lambrechts, P

2013-09-01

To provide a three-dimensional representation of external cervical resorption (ECR) with microscopy, stereo microscopy, cone beam computed tomography (CT), microfocus CT and scanning electron microscopy (SEM). External cervical resorption is an aggressive form of root resorption, leading to a loss of dental hard tissues. This is due to clastic action, activated by a damage of the covering cementum and stimulated probably by infection. Clinically, it is a challenging situation as it is characterized by a late symptomatology. This is due to the pericanalar protection from a resorption-resistant sheet, composed of pre-dentine and surrounding dentine. The clastic activity is often associated with an attempt to repair, seen by the formation of osteoid tissue. Cone beam CT is extremely useful in the diagnoses and treatment planning of ECR. SEM analyses provide a better insight into the activity of osteoclasts. The root canal is surrounded by a layer of dentine that is resistant to resorption. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Characterization of Antisticking Layers for UV Nanoimprint Lithography Molds with Scanning Probe Microscopy

NASA Astrophysics Data System (ADS)

Masaaki Kurihara,; Sho Hatakeyama,; Noriko Yamada,; Takeya Shimomura,; Takaharu Nagai,; Kouji Yoshida,; Tatsuya Tomita,; Morihisa Hoga,; Naoya Hayashi,; Hiroyuki Ohtani,; Masamichi Fujihira,

2010-06-01

Antisticking layers (ASLs) on UV nanoimprint lithography (UV-NIL) molds were characterized by scanning probe microscopies (SPMs) in addition to macroscopic analyses of work of adhesion and separation force. Local physical properties of the ASLs were measured by atomic force microscopy (AFM) and friction force microscopy (FFM). The behavior of local adhesive forces measured with AFM on several surfaces was consistent with that of work of adhesion obtained from contact angle. The ASLs were coated by two different processes, i.e., one is a vapor-phase process and the other a spin-coating process. The homogeneity of the ASLs prepared by the vapor-phase process was better than that of those prepared by the spin-coating process. In addition, we measured the thicknesses of ASL patterns prepared by a lift-off method to investigate the effect of the ASL thicknesses on critical dimensions of the molds with ASLs and found that this effect is not negligible.

Microscopy as a statistical, Rényi-Ulam, half-lie game: a new heuristic search strategy to accelerate imaging.

PubMed

Drumm, Daniel W; Greentree, Andrew D

2017-11-07

Finding a fluorescent target in a biological environment is a common and pressing microscopy problem. This task is formally analogous to the canonical search problem. In ideal (noise-free, truthful) search problems, the well-known binary search is optimal. The case of half-lies, where one of two responses to a search query may be deceptive, introduces a richer, Rényi-Ulam problem and is particularly relevant to practical microscopy. We analyse microscopy in the contexts of Rényi-Ulam games and half-lies, developing a new family of heuristics. We show the cost of insisting on verification by positive result in search algorithms; for the zero-half-lie case bisectioning with verification incurs a 50% penalty in the average number of queries required. The optimal partitioning of search spaces directly following verification in the presence of random half-lies is determined. Trisectioning with verification is shown to be the most efficient heuristic of the family in a majority of cases.

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

PubMed Central

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

2010-01-01

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

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

PubMed

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

A high-temperature superconducting Helmholtz probe for microscopy at 9.4 T.

PubMed

Hurlston, S E; Brey, W W; Suddarth, S A; Johnson, G A

1999-05-01

The design and operation of a high-temperature superconducting (HTS) probe for magnetic resonance microscopy (MRM) at 400 MHz are presented. The design of the probe includes a Helmholtz coil configuration and a stable open-cycle cooling mechanism. Characterization of coil operating parameters is presented to demonstrate the suitability of cryo-cooled coils for MRM. Specifically, the performance of the probe is evaluated by comparison of signal-to-noise (SNR) performance with that of a copper Helmholtz pair, analysis of B1 field homogeneity, and quantification of thermal stability. Images are presented to demonstrate the SNR advantage of the probe for typical MRM applications.

A low-cost photoacoustic microscopy system with a laser diode excitation

PubMed Central

Wang, Tianheng; Nandy, Sreyankar; Salehi, Hassan S.; Kumavor, Patrick D.; Zhu, Quing

2014-01-01

Photoacoustic microscopy (PAM) is capable of mapping microvasculature networks in biological tissue and has demonstrated great potential for biomedical applications. However, the clinical application of the PAM system is limited due to the use of bulky and expensive pulsed laser sources. In this paper, a low-cost optical-resolution PAM system with a pulsed laser diode excitation has been introduced. The lateral resolution of this PAM system was estimated to be 7 µm by imaging a carbon fiber. The phantoms made of polyethylene tubes filled with blood and a mouse ear were imaged to demonstrate the feasibility of this PAM system for imaging biological tissues. PMID:25401019

“Deterministic” quantum plasmonics.

PubMed

Cuche, Aurélien; Mollet, Oriane; Drezet, Aurélien; Huant, Serge

2010-11-10

We demonstrate “deterministic” launching of propagative quantum surface-plasmon polaritons at freely chosen positions on gold plasmonic receptacles. This is achieved by using as a plasmon launcher a near-field scanning optical source made of a diamond nanocrystal with two nitrogen-vacancy color-center occupancy. Our demonstration relies on leakage-radiation microscopy of a thin homogeneous gold film and on near-field optical microscopy of a nanostructured thick gold film. Our work paves the way to future fundamental studies and applications in quantum plasmonics that require an accurate positioning of single-plasmon sources and may open a new branch in plasmonics and nanophotonics, namely scanning quantum plasmonics.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

NASA Astrophysics Data System (ADS)

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-11-01

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue

NASA Astrophysics Data System (ADS)

Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Seltzer, Emily; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

2013-06-01

Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

PubMed

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-11-25

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

PubMed Central

Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-01-01

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format. PMID:27886235