Cell Isolation and Systems Analysis Related User Projects

• The Effects of Radiation Damage on Heteroepitaxial Interfaces
  Project Lead: Richard Kurtz
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  The interaction of radiation with materials controls the performance, reliability, and safety of conventional and advanced nuclear power systems. Energetic particles produced by nuclear reactions displace atoms in surrounding materials from their lattice sites, resulting in high local temperatures and formation of vacancies and interstitials that are deleterious to important material properties. Recent research suggests that nanospaced internal interfaces are powerful sinks for vacancies and interstitials [1-3]. Revolutionary improvements in radiation tolerance may be attainable if methods can be found to manipulate interface structures at the nanoscale to tailor their properties for optimal interface stability and point defect recombination, and to serve as traps for gaseous transmutants such as helium and hydrogen [4-6]. Interfaces contain miscoordinated atoms and excess volume that can assist recombination processes. A high-density of nanoscale features will dramatically increase interface area and shorten the diffusion path for defect recombination. Although recent experiments and modeling demonstrate the efficacy of this concept [7-9], the exact roles of interface free volume and the specific type of interface are not well understood. More importantly, an accurate physical description of point defect absorption and recombination processes occurring at interfaces does not exist. We propose to perform carefully controlled experiments utilizing well-characterized model interfaces to elucidate the fundamental mechanisms governing defect absorption and recombination at interfaces. The key scientific objectives of this research are 1) to understand how interface character affects absorption and recombination of radiation-induced defects, 2) to determine the ability of interfaces to delocalize radiation-induced defects to promote recombination, and 3) to determine the stability and evolution of interfaces under irradiation to high doses.
• Use of synthetic biology to probe molecular machines in photosynthesis
  Project Lead: Himadri Pakrasi
  Project Lead Institution: Washington University in St. Louis
  Abstract
  We have shown in our previous EMSL collaborations our ability to develop systems-­level models of cyanobacterial processes. In the present proposal, our objective is to characterize and improve the molecular machines in photosynthesis that govern bioenergy production in cyanobacteria. This proposal results from an EMSL workshop (MBGC 2.0) held in spring 2011. We propose to apply sets of experiments and analysis to achieve this goal using sophisticated imaging instrumentation available at EMSL. The profound expertise of the External Project Team in the areas of cyanobacterial and systems biology will be leveraged to attain the goals under this objective. The PIs Pakrasi, Sherman and Aurora have had extensive experience in collaborating with key investigators at EMSL/PNNL over the past 5 years, resulting in numerous publications (8-­15, as examples). The experiments outlined in this proposal will strive to investigate the products of a synthetic biology approach targeted in the following important areas of cyanobacterial biology; the goal of each is designed to provide new approaches to increasing photosynthetic productivity: 1) Photoshynthetic antenna modification; 2) photosynthetic electron transport and photosystem stoichiometry; 3) topology of photosynthetic membranes; and 4) carboxysome design and CO2 fixation.
• Characterization of Catalyst Materials in the Electron and Atom Probe Microscopes
  Project Lead: Ilke Arslan
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  The overall goal of this proposal is to develop a fundamental understanding of how to predict, synthesize, and control the compositions, structures, and catalytic function of supported metal clusters possessing designed atomic connectivity and ligand environments. Specifically, the PI of this proposal aims to advance the existing characterization tools at PNNL by combining several key tools in the electron and atom-probe microscopes. For example, the combination of atomic-resolution images of single or small metal cluster catalysts with 3-D electron tomography of its porous zeolite support for a complete understanding of the system connecting length and dimension scales; the combination of ex-situ or in-situ gas reduction combined with 3-D imaging to determine the 3-D distribution and morphology of the catalysts within the support before and after reduction; or the combination of electron tomography (ET) with atom probe tomography (APT) at various stages of reduction, providing the new capability of understanding the 3-D morphology, distribution, and chemistry with atomic resolution and without artifacts (the artifacts can be removed through the 3-D correlation of the ET and APT data). Electron energy loss spectroscopy (EELS) will be a major part of each set of experiments as an understanding of the changes in bonding and electronic structure is imperative to a fundamental understanding of the system as a whole. Further, it is important to characterize the structures, intermediates, and catalytic reaction products at various stages of growth and design, so not only is it essential to combine the techniques, it is also necessary to examine the materials through a “time-lapse" of various stages of design.
• Hematopoietic Stem and Progenitor Cell Kinetics of Irradiated Minipigs
  Project Lead: Jordan Smith
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  There is a need for a well-characterized animal model to test efficacy and safety of developmental prophylactics and therapeutics for acute radiation exposure in humans. We propose to characterize the kinetic profile of hematopoietic stem and progenitor cells in bone marrow aspirates of irradiated minipigs. Cell-specific antigens will be labeled with fluorescently tagged antibodies, and cells will be quantified using flow cytometry at the Environmental Molecular Science Laboratory (EMSL). The kinetic profile of hematopoietic stem and progenitor cells will provide well-characterized biomarkers of acute radiation syndrome in the minipig.
• Fundamental Studies of Vehicle Emission Control Catalysts
  Project Lead: Charles Peden
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  The abatement of environmentally harmful NOx compounds (NO, NO2, and N2O) emitted from mobile or stationary power sources remains a challenging task for the catalysis community. In particular, conventional three-way catalysts used in the exhaust after treatment technologies of internal combustion engines prove ineffective when the engine is operated under highly oxidizing conditions (to achieve better fuel efficiency). The problem is daunting, since reduction chemistry (NOx to N2) has to be carried out under highly oxidizing conditions. Several approaches have been proposed for lean-NOx abatement, each of them with its own specific sets of problems. The two technologies that seem to have clear advantages among the processes proposed are the selective catalytic reduction either with hydrocarbons (HC-SCR) or with ammonia (NH3-SCR), and NOx storage reduction (NSR). For the NH3-SCR technology, transition metal (in particular Fe and Cu) ion-exchanged zeolite catalysts have shown high activity and N2 selectivity. NSR catalysts work under cyclic operation conditions: oxidation of NO to NO2 followed by storage as nitrates/nitrites on alkali or alkaline oxides under lean conditions, and subsequent reduction of the released NOx under rich conditions. To further advance these important exhaust emission technologies, we are carrying out several research programs, funded by DOE/Office of Energy Efficiency and Renewable Energy (EERE)/Vehicle Technologies Program (VT), involving both programs that include direct collaboration with industry partners (at GM, Ford and Cummins), and a more fundamental program. For all of these programs, we rely on the use of a wide array of state-of-the-art catalyst characterization facilities in the EMSL at PNNL.
• Correlative Imaging and Spectroscopy of Biological Systems and Structural Dynamics
  Project Lead: James Evans
  Project Lead Institution: Environmental Molecular Sciences Laboratory
  Abstract
  The overall objective of this project is to create an integrative capability for combining dynamic transmission electron microscopy with femtosecond x-ray diffraction, in situ and automated secondary ion mass spectrometry and atom probe tomography to interrogate the structure and dynamics of biological systems. We will develop, adapt, and employ these state-of-the-art approaches to observe biological systems across multiple platforms with various spatial, temporal, and chemical resolutions. Key scientific ideas underlying the proposal involve: (a) assessing diffract-and-destroy compatibility with micro- and nanosecond single-shot electron pulses and femtosecond x-ray pulses; (b) acquiring movies of biological processes in real-time including conformational changes upon ligand binding and assembly/disassembly mechanisms; (c) designing and fabricating novel in situ flow cells that are more versatile for cross-platform analysis with electrons, x-rays and ions; (d) adapting nano-secondary ion mass spectrometry for robust serial section analysis of cells and tissues to provide 3-D chemical information; and (e) establish methodology (sample preparation and parameter optimization) to accurately reconstruct data of biomaterials using atom probe tomography. While the femtosecond x-ray diffraction will be performed at the Linac Coherent Light Source in California (already awarded 60 hours of LCLS beam time in 2013), all other instruments necessary for the success of this research are located at EMSL. The outcome of this research will be a centralized methodology that couples an array of advanced instrumentation techniques to obtain high spatial, chemical, and temporal resolution data of biological processes. The planned integration of imaging and spectroscopy platforms will yield a novel capability at EMSL for true multiscale and multimodal analysis and will enable a better understanding of many biological systems including biofilm organization, enzymatic energy transduction, and epithelial cell interactions with nanomaterial. Furthermore, the research will result in high-quality journal publications of both fundamental knowledge and state-of-the-art imaging technologies using facilities and equipment available at Environmental Molecular Sciences Laboratory.
• Quantifying selective toxicity of nanoparticles in zebrafish xenograft through advanced microscopy
  Project Lead: Robert Tanguay
  Project Lead Institution: Oregon State University
  Abstract
  Zinc oxide nanoparticles (ZnO-NPs) possess a 28-35 fold preferential cytotoxicity toward cancerous T cells in culture. We hypothesize that this cell dependent selective cytotoxicity extends to other metal oxide NPs. However, confirmation of whether this cell specific cytotoxicity persists in vivo remains to be seen. Zebrafish are a well-established model for studying developmental biology and investigating interactions of NPs with living systems at multiple levels of biological organization. Their transparency and small size make this model adaptable to rapid through-put, whole animal investigations. Through the use of an innovative zebrafish xenograft model, we can efficiently identify the properties of NPs that reduce adverse effects to the zebrafish while investigating the traits that enhance their selectivity toward cancer. Access to EMSL facilities will help establish the efficacy of the preferential toxicity of the ZnO-NPs by microscopic imaging and quantification of the xenotransplant size and distribution before and after treatment with the NPs. Imaging of xenografts exposed to FITC encapsulated ZnO-NPs will allow for measuring uptake, distribution, and potential NP-biological interactions. With this proposal, we will help fulfill EMSL’s Science of Interfacial Phenomena theme with a focus on the interactions of nanomaterials with living systems. This will help provide new design and synthesis rules for producing safer nanomaterials while maintaining critical performance properties.
• Interface-Specific Radiation Induced Segregation in Nanocrystalline Alloys
  Project Lead: Mitra Taheri
  Project Lead Institution: Drexel University
  Abstract
  The aim of the proposed project is to provide a fundamental understanding of how metallic nanocrystalline alloys can be utilized as radiation tolerant materials in future nuclear reactors. This aim will be achieved by utilizing the versatility of interfacial analysis tools in PNNL’s EMSL in conjunction with nanomaterial synthesis capabilities in CINT-LANL, in-situ TEM capabilities at both Drexel University and Argonne National Laboratory, and multi scale simulation expertise at PNNL. Although much is known already about the effects of radiation damage accumulation, information about its origin and mechanisms at the nanoscale and how these mechanisms evolve as a function of grain boundary (GB) structure/character and density (i.e., grain size) is still unclear. Research oriented towards understanding the fundamentals of damage mechanism of materials under extreme environments was identified as an important priority research direction for basic research related to advanced energy technology by the Office of Basic Energy Sciences. Relevant to the EMSL proposal call, the proposed systematic and multiscale investigation of radiation damage as a function of GB type and GB density falls in line with the “Science of Interfacial Phenomena” science theme, including in-situ real-time investigations relevant to energy production. The proposed project is expected to contribute towards the development of predictive models of interfacial processes. The anticipated results will support the development of new, stronger, multifunctional, damage-tolerant materials for future energy applications.
• Interfacial and Multiphase Characterization of Next Generation Inorganic Resists Synthesized Through Sustainable Materials Chemistries
  Project Lead: Liney Arnadottir
  Project Lead Institution: Oregon State University
  Abstract
  Transformation of small molecular precursors into inorganic solid-state compounds via solution methods has mainly focused on sol-gel techniques involving hydrolysis and condensation reactions of metal-organic compounds in bulk solutions. This method generally produces porous structures because of the difficulties in controlling the hydrolysis and condensation reactions leading to sol-gel formation. As part of the National Science Foundation Center for Sustainable Materials Chemistry (CSMC), we have undertaken a revolutionary approach to forming high-quality inorganic multicomponent films where we trap reactive precursors in an aqueous-based thin film and then induce controlled condensation. Using these approaches we have synthesized smooth, fully dense, crack-free, high-quality films of many compositions with controlled thickness down to 8 Å, including HfO2, ZrO2, HfO2-x(SO4)x, ZrO2-x(SO4)x, and Al4O3(PO4)2. The functionality of the resulting materials has been borne out through fabrication of high-performance thin-film transistors and displays,[1,2] dielectric mirrors and band-pass filters,[3] diffraction and light-trapping gratings,[4,5] and leading-edge directly imaged nanostructures via electron-beam and extreme ultra-violet lithographies.[6,7] The condensation reactions for these films can be initiated through thermal, photon, and electron stimulated processes which has lead to advances in nanolaminate fabrication (with single-digit-nm layer thicknesses) and direct sub-10-nm nanopatterning. The objective of this proposal is to develop a fundamental understanding of interfacial reaction mechanisms for film formation, interdiffusion between nanolaminate layers, and the role of precursor chemistries on nanopatterning fidelity for next generation photoresists. This proposal is well aligned with the EMSL's Science Theme on the “Science of Interfacial Phenomena,” and the EMSL capabilities will allow in-situ integrated approaches that will combine both experimental and theoretical methods to advance sustainable materials chemistries relevant to energy efficient functional materials synthesis.
• Optimizing Oil Production in Oleaginous Yeast by Cell-Wide Measurements and Genome-Based Models
  Project Lead: Scott Baker
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  Develop tools for cell-wide measurement of metabolites and lipids, which, along with transcriptional data, will allow the construction of genome-scale metabolic models, as well as models of transcriptional regulation, that will guide the further metabolic engineering of Y. lipolytica. This engineering will aim at improving the following fermentation characteristics of the present strain, all important figures of merit in the development of a cost-effective process for the conversion of renewable resources to lipids for biofuel production: • Improve conversion yield of glucose to greater than 0.25 grams of oil per gram of glucose consumed • Improve total volumetric productivities to double of present values • Facilitate utilization of acetate as substrate for lipid production and increase the overall conversion yield to lipids, and, • Improve xylose assimilation and conversion to lipids.