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Oak Ridge National Laboratory

30 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Bacterial Immobilization for Imaging by Atomic Force Microscopy
David P. Allison 1,2, Claretta J. Sullivan 3, Ninell Pollas Mortensen 1,2, Scott T. Retterer 1,4, Mitchel Doktycz 1,4
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).

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Engineering

Fabrication of Spatially Confined Complex Oxides
Hangwen Guo 1,2, Thomas Z. Ward 1
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.

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Environment

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Colin W. Bell 1, Barbara E. Fricks 1, Jennifer D. Rocca 1, Jessica M. Steinweg 2, Shawna K. McMahon 3, Matthew D. Wallenstein 1
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.

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Biology

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity
Tingting Xu 1, Dan M. Close 2, James D. Webb 3, Steven A. Ripp 2,3, Gary S. Sayler 1,2,3
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.

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Bioengineering

Sealable Femtoliter Chamber Arrays for Cell-free Biology
Sarah Elizabeth Norred 1,2, Patrick M. Caveney 1,2, Scott T. Retterer 1,2, Jonathan B. Boreyko 1,2, Jason D. Fowlkes 2,3, Charles Patrick Collier 2, Michael L. Simpson 1,2,3
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.

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Biology

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers
Joseph S. Najem 1, Myles D. Dunlap 2, Anthony Yasmann 3, Eric C. Freeman 4, John W. Grant 5, Sergei Sukharev 3, Donald J. Leo 4
1Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, 2School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University, 3Department of Biology, University of Maryland, 4College of Engineering, University of Georgia, 5Department of Engineering Sciences and Mechanics, Virginia Polytechnic Institute and State University

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

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Chemistry

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering
Youngkyu Han 1, Suk-kyun Ahn 2,3, Zhe Zhang 1,4, Gregory S. Smith 1, Changwoo Do 1
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.

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JoVE Journal

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand
Charles A. Seipp 1,2, Neil J. Williams 1,3, Radu Custelcean 1
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.

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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 Guennoc 1, Christophe Rose 2, Frédéric Guinnet 1, Igor Miquel 1, Jessy Labbé 3, Aurélie Deveau 1
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.

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JoVE Journal

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform
Andrea C. Timm 1, Michelle C. Halsted 2, Jared L. Wilmoth 1, Scott T. Retterer 1,3
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.

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Bioengineering

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform
Jayde A. Aufrecht 1,2, Jennifer M. Ryan 3, Sahar Hasim 4, David P. Allison 2,3, Andreas Nebenführ 3, Mitchel J. Doktycz 1,2, Scott T. Retterer 1,2
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.

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Chemistry

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering
Brian Morgan 1, Samantha J. Rinehart 1, Mark D. Dadmun 1,2
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.

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Engineering

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers
Mohammadali Masigol 1, Niloy Barua 1, Bradley S. Lokitz 2, Ryan R. Hansen 1
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.

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Biochemistry

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling
Ryan C. Oliver 1, Swe-Htet Naing 2, Kevin L. Weiss 1, Sai Venkatesh Pingali 1, Raquel L. Lieberman 2, Volker S. Urban 1
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.

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JoVE Core

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes
Joseph S. Najem 1,2, Graham J. Taylor 2,3, Nick Armendarez 4, Ryan J. Weiss 5, Md Sakib Hasan 5, Garrett S. Rose 5, Catherine D. Schuman 6, Alex Belianinov 7, Stephen A. Sarles 2, C. Patrick Collier 2,3,7
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.

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Engineering

A Silicon-tipped Fiber-optic Sensing Platform with High Resolution and Fast Response
Guigen Liu *1, Qiwen Sheng *1, Weilin Hou 2, Matthew L. Reinke 3, Ming Han 1,4
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.

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Neuroscience

Computer-based Multitaper Spectrogram Program for Electroencephalographic Data
Christopher B. O'Brien 1, Helen A. Baghdoyan 1,2,3, Ralph Lydic 1,2,3
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.

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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. Meagher 6, Ryan D. Ross 6, Jean C. Bilheux 1,7, Singanallur Venkatakrishnan 8, Jiao Y.Y. Lin 1,9, Yuxuan Zhang 1, Matthew R. Pearson 9,10, Erik Stringfellow 1
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.

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Chemistry

Neutron Crystallography Data Collection and Processing for Modelling Hydrogen Atoms in Protein Structures
Gabriela C. Schröder 1,2, Flora Meilleur 1,2
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.

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Environment

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures
Jeremy R. Chen See 1,2, Olivia Wright 1, Lavinia V. Unverdorben 1,2, Nathan Heibeck 1, Stephen M. Techtmann 3, Terry C. Hazen 4,5, Regina Lamendella 1,2
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.

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Chemistry

Picometer-Precision Atomic Position Tracking through Electron Microscopy
Leixin Miao 1, Adrian Chmielewski 1, Debangshu Mukherjee 2, Nasim Alem 1
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).

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Engineering

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope
Kinga A. Unocic 1, Dale K. Hensley 1, Franklin S. Walden 2, Wilbur C. Bigelow 3, Michael B. Griffin 4, Susan E. Habas 4, Raymond R. Unocic 1, Lawrence F. Allard 5
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.

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Bioengineering

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
Jaime Lorenzo N. Dinglasan 1,3, David T. Reeves 2,3, Robert L. Hettich 3, Mitchel J. Doktycz 3
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.

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Neuroscience

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)
Nannan Zhang 1, Zhe Zhang 1,2, Ilker Ozden 2, Shinghua Ding 1,2
1Dalton Cardiovascular Research Center, University of Missouri-Columbia, 2Department of Biomedical, Biological and Chemical Engineering, University of Missouri-Columbia

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

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Medicine

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer
Chenhao Hu *1,2,3, Zhe Zhang *1,2,3, Lei Zhang 1,2,3, Ruihan Liu 2,3, Jun Yan 1,2,3, Qi Sun 1,2,3, Guanghui Wang 1, Junjun She 1,2,3
1Department of General Surgery, The First Affiliated Hospital of Xian Jiaotong University, 2Center for Gut Microbiome Research, Med-X Institute, The First Affiliated Hospital of Xian Jiao tong University, 3Department of High Talent, The First Affiliated Hospital of Xian Jiaotong University

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

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Engineering

Forming Micro-and Nano-Plastics from Agricultural Plastic Films for Employment in Fundamental Research Studies
Anton F. Astner 1, Douglas G. Hayes 1, Hugh M. O'Neill 2, Barbara R. Evans 3, Sai Venkatesh Pingali 2, Volker S. Urban 2, Timothy M. Young 4
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.

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Developmental Biology

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research
Yang Zhang *1,2,3, Zhe Zhang *1,2,3, Nannan Kang 1,2,3, Xiaoqiang Sheng 1,2,3
1Center for Reproductive Medicine and Obstetrics and Gynecology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, 2State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 3Center for Molecular Reproductive Medicine, Nanjing University

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

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Neutron Scattering in the Biological Sciences: Techniques And Applications
Flora Meilleur 1,2
1Department of Molecular and Structural Biochemistry, North Carolina State University, 2Neutron Scattering Division, Oak Ridge National Laboratory

Neutron Scattering in the Biological Sciences: Techniques And Applications

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State-of-the-Art Electron Microscopy For Physical Sciences Research
Michael J. Zachman 1
1Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

State-of-the-Art Electron Microscopy For Physical Sciences Research

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Bioengineering

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants
Jessica M. Morgan 1, Joanna Jelenska 2, Dale K. Hensley 3, Pengju Li 4, Bernadeta R. Srijanto 3, Scott T. Retterer 3,5, Robert F. Standaert 6, Jennifer L. Morrell-Falvey 5, Jean T. Greenberg 2
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.

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