Oak Ridge National Laboratory View Institution's Website 24 articles published in JoVE Bioengineering Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants Jessica M. Morgan1, Joanna Jelenska2, Dale K. Hensley3, Pengju Li4, Bernadeta R. Srijanto3, Scott T. Retterer3,5, Robert F. Standaert6, Jennifer L. Morrell-Falvey5, Jean T. Greenberg2 1Biophysical Sciences, The University of Chicago, 2Molecular Genetics and Cell Biology, The University of Chicago, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 4Pritzker School of Molecular Engineering, The University of Chicago, 5Biosciences Division, Oak Ridge National Laboratory, 6Department of Chemistry, East Tennessee State University Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery. Engineering Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies Anton F. Astner1, Douglas G. Hayes1, Hugh M. O'Neill2, Barbara R. Evans3, Sai Venkatesh Pingali2, Volker S. Urban2, Timothy M. Young4 1Department of Biosystems Engineering and Soil Science, University of Tennessee, 2Neutron Scattering, Oak Ridge National Laboratory, 3Chemical Sciences Divisions, Oak Ridge National Laboratory, 4Center for Renewable Carbon, The University of Tennessee We show the formation and dimensional characterization of micro- and nanoplastics (MPs and NPs, respectively) using a stepwise process of mechanical milling, grinding, and imaging analysis. Bioengineering Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems Jaime Lorenzo N. Dinglasan1,3, David T. Reeves2,3, Robert L. Hettich3, Mitchel J. Doktycz3 1Graduate School of Genome Science & Technology, University of Tennessee Knoxville, 2Bredesen Center for Interdisciplinary Research, University of Tennessee Knoxville, 3Biosciences Division, Oak Ridge National Laboratory The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems. Engineering Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope Kinga A. Unocic1, Dale K. Hensley1, Franklin S. Walden2, Wilbur C. Bigelow3, Michael B. Griffin4, Susan E. Habas4, Raymond R. Unocic1, Lawrence F. Allard5 1Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 2Protochips Inc., 3Department of Materials Science & Engineering, University of Michigan, 4Catalytic Carbon Transformation & Scale-up, National Renewable Energy Laboratory, 5Materials Science & Technology Division, Oak Ridge National Laboratory Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods. Chemistry Picometer-Precision Atomic Position Tracking through Electron Microscopy Leixin Miao1, Adrian Chmielewski1, Debangshu Mukherjee2, Nasim Alem1 1Department of Materials Science & Engineering, The Pennsylvania State University, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory This work presents a workflow for atomic position tracking in atomic resolution transmission electron microscopy imaging. This workflow is performed using an open-source Matlab app (EASY-STEM). Biology Neutron Radiography and Computed Tomography of Biological Systems at the Oak Ridge National Laboratory's High Flux Isotope Reactor Hassina Z. Bilheux*1, Maria Cekanova*2,3,4, Jeffrey M. Warren*5, Matthew J. Meagher6, Ryan D. Ross6, Jean C. Bilheux1,7, Singanallur Venkatakrishnan8, Jiao Y.Y. Lin1,9, Yuxuan Zhang1, Matthew R. Pearson9,10, Erik Stringfellow1 1Neutron Scattering Division, Oak Ridge National Laboratory, 2College of Veterinary Medicine, The University of Tennessee, 3UT-ORNL Graduate School of Genome, Science and Technology, The University of Tennessee, 4Integrity Laboratories, 5Environmental Sciences Division, Oak Ridge National Laboratory, 6Department of Cell & Molecular Medicine, Rush Medical College, Rush University, 7Computer Science and Mathematics Division, Oak Ridge National Laboratory, 8Electrification and Energy Infrastructures Division, Oak Ridge National Laboratory, 9Now at Second Target Station Project, Oak Ridge National Laboratory, 10Neutron Technologies Division, Oak Ridge National Laboratory This manuscript describes a protocol for neutron radiography and computed tomography of biological samples using a High Flux Isotope Reactor (HFIR) CG-1D beamline to measure a metal implant in a rat femur, a mouse lung, and an herbaceous plant root/soil system. Environment Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures Jeremy R. Chen See1,2, Olivia Wright1, Lavinia V. Unverdorben1,2, Nathan Heibeck1, Stephen M. Techtmann3, Terry C. Hazen4,5, Regina Lamendella1,2 1Department of Biology, Juniata College, 2Wright Labs, LLC, 3Department of Biological Sciences, Michigan Technological University, 4Biosciences Division, Oak Ridge National Laboratory, 5Department of Civil and Environmental Engineering, University of Tennessee Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities. Chemistry Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures Gabriela C. Schröder1,2, Flora Meilleur1,2 1Department of Molecular and Structural Biochemistry, North Carolina State University, 2Neutron Scattering Division, Oak Ridge National Laboratory Neutron protein crystallography is a structural technique that permits the localization of hydrogen atoms, thereby providing important mechanistic details of protein function. We present here the workflow for mounting a protein crystal, neutron diffraction data collection, structure refinement and analysis of the neutron scattering length density maps. Neuroscience Computer-based Multitaper Spectrogram Program for Electroencephalographic Data Christopher B. O'Brien1, Helen A. Baghdoyan1,2,3, Ralph Lydic1,2,3 1Department of Psychology, University of Tennessee, 2Department of Anesthesiology, University of Tennessee, 3Oak Ridge National Laboratory This protocol provides an open source, compiled MATLAB program that generates multitaper spectrograms for electroencephalographic data. Bioengineering Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes Joseph S. Najem1,2, Graham J. Taylor2,3, Nick Armendarez4, Ryan J. Weiss5, Md Sakib Hasan5, Garrett S. Rose5, Catherine D. Schuman6, Alex Belianinov7, Stephen A. Sarles2, C. Patrick Collier2,3,7 1Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, 3Bredesen Center for Interdisciplinary Research, University of Tennessee, 4Department of Biosystems and Agriculture Engineering, University of Kentucky, 5Department of Electrical Engineering and Computer Science, University of Tennessee, 6Computer Science and Mathematics Division, Oak Ridge National Laboratory, 7Center for Nanophase Materials Sciences, Oak Ridge National Laboratory Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane. Engineering A Silicon-tipped Fiber-optic Sensing Platform with High Resolution and Fast Response Guigen Liu*1, Qiwen Sheng*1, Weilin Hou2, Matthew L. Reinke3, Ming Han1,4 1Department of Electrical and Computer Engineering, Michigan State University, 2Naval Research Laboratory, Stennis Space Center, 3Oak Ridge National Laboratory, 4Department of Electrical and Computer Engineering, University of Nebraska-Lincoln This work reports an innovative silicon-tipped fiber-optic sensing platform (Si-FOSP) for high-resolution and fast-response measurement of a variety of physical parameters, such as temperature, flow, and radiation. Applications of this Si-FOSP span from oceanographic research, mechanical industry, to fusion energy research. Biochemistry Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling Ryan C. Oliver1, Swe-Htet Naing2, Kevin L. Weiss1, Sai Venkatesh Pingali1, Raquel L. Lieberman2, Volker S. Urban1 1Neutron Scattering Division, Oak Ridge National Laboratory, 2School of Chemistry and Biochemistry, Georgia Institute of Technology This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent. Engineering Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers Mohammadali Masigol1, Niloy Barua1, Bradley S. Lokitz2, Ryan R. Hansen1 1Chemical Engineering Department, Kansas State University, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity. Chemistry Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering Brian Morgan1, Samantha J. Rinehart1, Mark D. Dadmun1,2 1Department of Chemistry, University of Tennessee, Knoxville, 2Chemical Sciences Division, Oak Ridge National Laboratory A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented. Bioengineering Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform Jayde A. Aufrecht1,2, Jennifer M. Ryan3, Sahar Hasim4, David P. Allison2,3, Andreas Nebenführ3, Mitchel J. Doktycz1,2, Scott T. Retterer1,2 1Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, 2Bioscience Division and Center for Nanophase Materials Sciences, Oak Ridge national Laboratory, 3Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 4Department of Microbiology, University of Tennessee This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means. Bioengineering Assembly and Tracking of Microbial Community Development within a Microwell Array Platform Andrea C. Timm1, Michelle C. Halsted2, Jared L. Wilmoth1, Scott T. Retterer1,3 1Biosciences Division, Oak Ridge National Laboratory, 2Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated. Immunology and Infection A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy Cora Miquel Guennoc1, Christophe Rose2, Frédéric Guinnet1, Igor Miquel1, Jessy Labbé3, Aurélie Deveau1 1Interactions Arbres – Microorganismes, UMR1136, INRA Université de Lorraine, 2Ecologie et Ecophysiologie Forestières - PTEF, UMR 1137, INRA Université de Lorraine, 3Biosciences Division, Oak Ridge National Laboratory This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy. Chemistry Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand Charles A. Seipp1,2, Neil J. Williams1,3, Radu Custelcean1 1Chemical Sciences Division, Oak Ridge National Laboratory, 2Department of Chemistry, The University of Texas at Austin, 3Department of Chemistry, The University of Tennessee A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented. Chemistry Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering Youngkyu Han1, Suk-kyun Ahn2,3, Zhe Zhang1,4, Gregory S. Smith1, Changwoo Do1 1Biology and Soft Matter Division, Neutron Science Directorate, Oak Ridge National Laboratory, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Polymer Science and Engineering, Pusan National University, 4Jülich Center for Neutron Science, Forschungszentrum Jülich A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented. Bioengineering Sealable Femtoliter Chamber Arrays for Cell-free Biology Sarah Elizabeth Norred1,2, Patrick M. Caveney1,2, Scott T. Retterer1,2, Jonathan B. Boreyko1,2, Jason D. Fowlkes2,3, Charles Patrick Collier2, Michael L. Simpson1,2,3 1Bredesen Center, University of Tennessee, Knoxville, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, 3Department of Materials Science and Engineering, University of Tennessee, Knoxville A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression. Environment High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities Colin W. Bell1, Barbara E. Fricks1, Jennifer D. Rocca1, Jessica M. Steinweg2, Shawna K. McMahon3, Matthew D. Wallenstein1 1Natural Resource Ecology Laboratory, Colorado State University, 2Biosciences Division, Oak Ridge National Laboratory, 3Department of Bioengineering, University of Colorado To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation. Biology Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity Tingting Xu1, Dan M. Close2, James D. Webb3, Steven A. Ripp2,3, Gary S. Sayler1,2,3 1The Joint Institute for Biological Sciences, Oak Ridge National Laboratory, 2490 BioTech, Inc., 3The Center for Environmental Biotechnology, The University of Tennessee, Knoxville Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation. Engineering Fabrication of Spatially Confined Complex Oxides Hangwen Guo1,2, Thomas Z. Ward1 1Materials Science and Technology Division, Oak Ridge National Laboratory, 2Department of Physics & Astronomy, University of Tennessee, Knoxville We describe the use of pulsed laser deposition (PLD), photolithography and wire-bonding techniques to create micrometer scale complex oxides devices. The PLD is utilized to grow epitaxial thin films. Photolithography and wire-bonding techniques are introduced to create practical devices for measurement purposes. Bioengineering Bacterial Immobilization for Imaging by Atomic Force Microscopy David P. Allison1,2, Claretta J. Sullivan3, Ninell Pollas Mortensen1,2, Scott T. Retterer1,4, Mitchel Doktycz1,4 1Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, 3Department of Surgery, Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).