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An Investigation on the Persistence of Uranium Hydride during Storage of Simulant Nuclear Waste Packages.
PUBLISHED: 07-16-2015
Synchrotron X-rays have been used to study the oxidation of uranium and uranium hydride when encapsulated in grout and stored in de-ionised water for 10 months. Periodic synchrotron X-ray tomography and X-ray powder diffraction have allowed measurement and identification of the arising corrosion products and the rates of corrosion. The oxidation rates of the uranium metal and uranium hydride were slower than empirically derived rates previously reported for each reactant in an anoxic water system, but without encapsulation in grout. This was attributed to the grout acting as a physical barrier limiting the access of oxidising species to the uranium surface. Uranium hydride was observed to persist throughout the 10 month storage period and industrial consequences of this observed persistence are discussed.
Authors: Rachel Pflieger, Tony Chave, Matthieu Virot, Sergey I. Nikitenko.
Published: 04-11-2014
The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.
21 Related JoVE Articles!
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Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability
Authors: Jodi R. Schilz, K. J. Reddy, Sreejayan Nair, Thomas E. Johnson, Ronald B. Tjalkens, Kem P. Krueger, Suzanne Clark.
Institutions: University of New Mexico, University of Wyoming, University of Wyoming, Colorado State University, Colorado State University, California Northstate University.
In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.
Environmental Sciences, Issue 100, Energy production, uranium in situ recovery, water decontamination, nanoparticles, toxicity, cytotoxicity, in vitro cell culture
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The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes
Authors: Andreas Phanopoulos, Nicholas Long, Philip Miller.
Institutions: Imperial College London.
Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-κ3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-κ3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-κ3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-κ3P{CH3CO(CH2)2CO2H}-κ2O](PF6) (6).
Chemistry, Issue 98, ligand, phosphine, coordination, complex, catalysis, ruthenium, biomass, levulinic acid
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Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Authors: Michael Heethoff, Lukas Helfen, Peter Cloetens.
Institutions: University of Tubingen, European Synchrotron Radiation Facility.
Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning. We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.
Developmental Biology, Issue 15, Synchrotron X-ray tomography, Acari, Oribatida, micro-arthropods, non-invasive investigation
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Flame Experiments at the Advanced Light Source: New Insights into Soot Formation Processes
Authors: Nils Hansen, Scott A. Skeen, Hope A. Michelsen, Kevin R. Wilson, Katharina Kohse-Höinghaus.
Institutions: Sandia National Laboratories, Lawrence Berkeley National Laboratory, Universität Bielefeld.
The following experimental protocols and the accompanying video are concerned with the flame experiments that are performed at the Chemical Dynamics Beamline of the Advanced Light Source (ALS) of the Lawrence Berkeley National Laboratory1-4. This video demonstrates how the complex chemical structures of laboratory-based model flames are analyzed using flame-sampling mass spectrometry with tunable synchrotron-generated vacuum-ultraviolet (VUV) radiation. This experimental approach combines isomer-resolving capabilities with high sensitivity and a large dynamic range5,6. The first part of the video describes experiments involving burner-stabilized, reduced-pressure (20-80 mbar) laminar premixed flames. A small hydrocarbon fuel was used for the selected flame to demonstrate the general experimental approach. It is shown how species’ profiles are acquired as a function of distance from the burner surface and how the tunability of the VUV photon energy is used advantageously to identify many combustion intermediates based on their ionization energies. For example, this technique has been used to study gas-phase aspects of the soot-formation processes, and the video shows how the resonance-stabilized radicals, such as C3H3, C3H5, and i-C4H5, are identified as important intermediates7. The work has been focused on soot formation processes, and, from the chemical point of view, this process is very intriguing because chemical structures containing millions of carbon atoms are assembled from a fuel molecule possessing only a few carbon atoms in just milliseconds. The second part of the video highlights a new experiment, in which an opposed-flow diffusion flame and synchrotron-based aerosol mass spectrometry are used to study the chemical composition of the combustion-generated soot particles4. The experimental results indicate that the widely accepted H-abstraction-C2H2-addition (HACA) mechanism is not the sole molecular growth process responsible for the formation of the observed large polycyclic aromatic hydrocarbons (PAHs).
Physics, Issue 87, Combustion, Flame, Energy Conversion, Mass Spectrometry, Photoionization, Synchrotron, Hydrocarbon, Soot, Aerosol, Isomer
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In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
Authors: William R. Brant, Siegbert Schmid, Guodong Du, Helen E. A. Brand, Wei Kong Pang, Vanessa K. Peterson, Zaiping Guo, Neeraj Sharma.
Institutions: University of Sydney, University of Wollongong, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, University of Wollongong, University of New South Wales.
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2 However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3 This research is challenging, and we outline a method to address these challenges using in situ NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries. We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the ‘roll-over’ cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ NPD experiment and initial directions are presented on how to analyze such complex in situ data.
Physics, Issue 93, In operando, structure-property relationships, electrochemical cycling, electrochemical cells, crystallography, battery performance
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Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models
Authors: Teresa E. Lever, Sabrina M. Braun, Ryan T. Brooks, Rebecca A. Harris, Loren L. Littrell, Ryan M. Neff, Cameron J. Hinkel, Mitchell J. Allen, Mollie A. Ulsas.
Institutions: University of Missouri, University of Missouri, University of Missouri.
This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.
Medicine, Issue 97, mouse, murine, rodent, swallowing, deglutition, dysphagia, videofluoroscopy, radiation, iohexol, barium, palatability, taste, translational, disease models
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Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities
Authors: Stephan Böttcher, Joachim Thiem.
Institutions: University of Hamburg.
Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zemplén.
Chemistry, Issue 99, Carbohydrates, Indoxyl-Glycosides, Indoxylic Acid Esters, Decarboxylation, Dyes/Pigments, Enzyme Activity, Nitrogen Heterocycles
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Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition
Authors: Rafael Jaramillo, Vera Steinmann, Chuanxi Yang, Katy Hartman, Rupak Chakraborty, Jeremy R. Poindexter, Mariela Lizet Castillo, Roy Gordon, Tonio Buonassisi.
Institutions: Massachusetts Institute of Technology, Massachusetts Institute of Technology, Harvard University, Massachusetts Institute of Technology, Harvard University.
Tin sulfide (SnS) is a candidate absorber material for Earth-abundant, non-toxic solar cells. SnS offers easy phase control and rapid growth by congruent thermal evaporation, and it absorbs visible light strongly. However, for a long time the record power conversion efficiency of SnS solar cells remained below 2%. Recently we demonstrated new certified record efficiencies of 4.36% using SnS deposited by atomic layer deposition, and 3.88% using thermal evaporation. Here the fabrication procedure for these record solar cells is described, and the statistical distribution of the fabrication process is reported. The standard deviation of efficiency measured on a single substrate is typically over 0.5%. All steps including substrate selection and cleaning, Mo sputtering for the rear contact (cathode), SnS deposition, annealing, surface passivation, Zn(O,S) buffer layer selection and deposition, transparent conductor (anode) deposition, and metallization are described. On each substrate we fabricate 11 individual devices, each with active area 0.25 cm2. Further, a system for high throughput measurements of current-voltage curves under simulated solar light, and external quantum efficiency measurement with variable light bias is described. With this system we are able to measure full data sets on all 11 devices in an automated manner and in minimal time. These results illustrate the value of studying large sample sets, rather than focusing narrowly on the highest performing devices. Large data sets help us to distinguish and remedy individual loss mechanisms affecting our devices.
Engineering, Issue 99, Solar cells, thin films, thermal evaporation, atomic layer deposition, annealing, tin sulfide
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Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Authors: Matthew Rames, Yadong Yu, Gang Ren.
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
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Culturing and Maintaining Clostridium difficile in an Anaerobic Environment
Authors: Adrianne N. Edwards, Jose M. Suárez, Shonna M. McBride.
Institutions: Emory University School of Medicine.
Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.
Immunology, Issue 79, Genetics, Bacteria, Anaerobic, Gram-Positive Endospore-Forming Rods, Spores, Bacterial, Gram-Positive Bacterial Infections, Clostridium Infections, Bacteriology, Clostridium difficile, Gram-positive, anaerobic chamber, spore, culturing, maintenance, cell culture
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Aseptic Laboratory Techniques: Plating Methods
Authors: Erin R. Sanders.
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: ● Perform plating procedures without contaminating media. ● Isolate single bacterial colonies by the streak-plating method. ● Use pour-plating and spread-plating methods to determine the concentration of bacteria. ● Perform soft agar overlays when working with phage. ● Transfer bacterial cells from one plate to another using the replica-plating procedure. ● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
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Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Authors: Elvira Romero, Reeder Robinson, Pablo Sobrado.
Institutions: Virginia Polytechnic Institute and State University.
Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6
Bioengineering, Issue 61, Stopped-flow, kinetic mechanism, SidA, C4a-hydroperoxyflavin, monooxygenase, Aspergillus fumigatus
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Flexural Rigidity Measurements of Biopolymers Using Gliding Assays
Authors: Douglas S. Martin, Lu Yu, Brian L. Van Hoozen.
Institutions: Lawrence University.
Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9. Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL. This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.
Biophysics, Issue 69, Bioengineering, Physics, Molecular Biology, Cellular Biology, microtubule, persistence length, flexural rigidity, gliding assay, mechanics, cytoskeleton, actin
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Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature
Authors: Andrew J. McElrone, Brendan Choat, Dilworth Y. Parkinson, Alastair A. MacDowell, Craig R. Brodersen.
Institutions: U.S. Department of Agriculture, University of California - Davis, University of Western Sydney, Lawrence Berkeley National Lab, University of Florida .
High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods. Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.
Plant Biology, Issue 74, Cellular Biology, Molecular Biology, Biophysics, Structural Biology, Physics, Environmental Sciences, Agriculture, botany, environmental effects (biological, animal and plant), plants, radiation effects (biological, animal and plant), CT scans, advanced visualization techniques, xylem networks, plant vascular function, synchrotron, x-ray micro-tomography, ALS 8.3.2, xylem, phloem, tomography, imaging
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Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
Authors: Jean-Marc Taymans, Fangye Gao, Veerle Baekelandt.
Institutions: KU Leuven and Leuven Institute for Neuroscience and Disease (LIND).
Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.
Cellular Biology, Issue 79, biology (general), biochemistry, bioengineering (general), LRRK1, LRRK2, metabolic labeling, 32P orthophosphate, immunoprecipitation, autoradiography
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Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Authors: Marca M. Doeff, Guoying Chen, Jordi Cabana, Thomas J. Richardson, Apurva Mehta, Mona Shirpour, Hugues Duncan, Chunjoong Kim, Kinson C. Kam, Thomas Conry.
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g. changes in redox states and bond lengths). In situ experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ experiments (e.g. on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ and in situ samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
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Synthesis and Microdiffraction at Extreme Pressures and Temperatures
Authors: Barbara Lavina, Przemyslaw Dera, Yue Meng.
Institutions: University of Nevada, Las Vegas, University of Chicago, Carnegie Institution of Washington.
High pressure compounds and polymorphs are investigated for a broad range of purposes such as determine structures and processes of deep planetary interiors, design materials with novel properties, understand the mechanical behavior of materials exposed to very high stresses as in explosions or impacts. Synthesis and structural analysis of materials at extreme conditions of pressure and temperature entails remarkable technical challenges. In the laser heated diamond anvil cell (LH-DAC), very high pressure is generated between the tips of two opposing diamond anvils forced against each other; focused infrared laser beams, shined through the diamonds, allow to reach very high temperatures on samples absorbing the laser radiation. When the LH-DAC is installed in a synchrotron beamline that provides extremely brilliant x-ray radiation, the structure of materials under extreme conditions can be probed in situ. LH-DAC samples, although very small, can show highly variable grain size, phase and chemical composition. In order to obtain the high resolution structural analysis and the most comprehensive characterization of a sample, we collect diffraction data in 2D grids and combine powder, single crystal and multigrain diffraction techniques. Representative results obtained in the synthesis of a new iron oxide, Fe4O5 1 will be shown.
Physics, Issue 80, x-ray diffraction, geochemistry, geophysics, solid-state physics, high-pressure, high-temperature, Diamond anvil cell, micro-diffraction, novel materials, iron oxides, mantle mineralogy
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Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies
Authors: Katla Sai Krishna, Sanchita Biswas, Chelliah V. Navin, Dawit G. Yamane, Jeffrey T. Miller, Challa S.S.R. Kumar.
Institutions: Louisiana State University, Louisiana State University, Louisiana State University, Argonne National Laboratory.
Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.
Bioengineering, Issue 81, Millifluidics, Millifluidic Device, Time-resolved Kinetics, Synthesis, Catalysis, Nanomaterials, Lab-on-a-Chip
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Supercritical Nitrogen Processing for the Purification of Reactive Porous Materials
Authors: Nicholas P. Stadie, Elsa Callini, Philippe Mauron, Andreas Borgschulte, Andreas Züttel.
Institutions: Empa, Swiss Federal Laboratories for Materials Science and Technology.
Supercritical fluid extraction and drying methods are well established in numerous applications for the synthesis and processing of porous materials. Herein, nitrogen is presented as a novel supercritical drying fluid for specialized applications such as in the processing of reactive porous materials, where carbon dioxide and other fluids are not appropriate due to their higher chemical reactivity. Nitrogen exhibits similar physical properties in the near-critical region of its phase diagram as compared to carbon dioxide: a widely tunable density up to ~1 g ml-1, modest critical pressure (3.4 MPa), and small molecular diameter of ~3.6 Å. The key to achieving a high solvation power of nitrogen is to apply a processing temperature in the range of 80-150 K, where the density of nitrogen is an order of magnitude higher than at similar pressures near ambient temperature. The detailed solvation properties of nitrogen, and especially its selectivity, across a wide range of common target species of extraction still require further investigation. Herein we describe a protocol for the supercritical nitrogen processing of porous magnesium borohydride.
Chemistry, Issue 99, supercritical, purification, drying, extraction, solvent, nitrogen, activation, microporous materials, surface area, reactive materials, complex hydrides, hydrogen storage
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Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software
Authors: Simon R. Stockwell, Sibylle Mittnacht.
Institutions: UCL Cancer Institute.
Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators. Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1 to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.
Cellular Biology, Issue 94, Image analysis, High-content analysis, Screening, Microscopy, Individual cell analysis, Multiplexed assays
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Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology
Authors: William S. Phipps, Zhizhong Yin, Candice Bae, Julia Z. Sharpe, Andrew M. Bishara, Emily S. Nelson, Aaron S. Weaver, Daniel Brown, Terri L. McKay, DeVon Griffin, Eugene Y. Chan.
Institutions: DNA Medicine Institute, Harvard Medical School, NASA Glenn Research Center, ZIN Technologies.
Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.
Cellular Biology, Issue 93, Point-of-care, prototype, diagnostics, spaceflight, reduced gravity, parabolic flight, flow cytometry, fluorescence, cell counting, micromixing, spiral-vortex, blood mixing
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.