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Lawrence Berkeley National Laboratory

33 ARTICLES PUBLISHED IN JoVE

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Biology

Label-free in situ Imaging of Lignification in Plant Cell Walls
Martin Schmidt 1, Pradeep Perera 1, Adam M. Schwartzberg 2, Paul D. Adams 3, P. James Schuck 2
1Energy Biosciences Institute, University of California, Berkeley, 2Molecular Foundry, Lawrence Berkeley National Laboratory, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

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Bioengineering

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
Helen Tran 1, Sarah L. Gael 1, Michael D. Connolly 1, Ronald N. Zuckermann 1
1Molecular Foundry, Lawrence Berkeley National Laboratory

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

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Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)
Chun-Han Lin 1,2, Jonathan K. Lee 1, Mark A. LaBarge 1
1Life Science Division, Lawrence Berkeley National Laboratory, 2Department of Comparative Biochemistry, University of California, Berkeley

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

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Medicine

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture
Mark A. LaBarge 1, James C. Garbe 1, Martha R. Stampfer 1
1Life Science Division, Lawrence Berkeley National Laboratory

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

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Engineering

Revealing Dynamic Processes of Materials in Liquids Using Liquid Cell Transmission Electron Microscopy
Kai-Yang Niu 1, Hong-Gang Liao 1, Haimei Zheng 1
1Materials Sciences Division, Lawrence Berkeley National Laboratory

We have developed a self-contained liquid cell, which allows imaging through liquids using a transmission electron microscope. Dynamic processes of nanoparticles in liquids can be revealed in real time with sub-nanometer resolution.

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JoVE Journal

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature
Andrew J. McElrone 1,2, Brendan Choat 3, Dilworth Y. Parkinson 4, Alastair A. MacDowell 4, Craig R. Brodersen 5
1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

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Engineering

Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation
Amir Golan 1, Musahid Ahmed 1
1Chemical Sciences Division, Lawrence Berkeley National Laboratory

A molecular beam coupled to tunable vacuum ultraviolet photoionization mass spectrometer at a synchrotron provides a convenient tool to explore the electronic structure of isolated gas phase molecules and clusters. Proton transfer mechanisms in DNA base dimers were elucidated with this technique.

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Engineering

Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Marca M. Doeff 1, Guoying Chen 1, Jordi Cabana 1,2, Thomas J. Richardson 1, Apurva Mehta 3, Mona Shirpour 1, Hugues Duncan 1, Chunjoong Kim 1, Kinson C. Kam 4, Thomas Conry 5
1Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory, 2Department of Chemistry, University of Illinois at Chicago, 3Stanford Synchrotron Radiation Lightsource, 4Haldor Topsøe A/S, 5PolyPlus Battery Company

We describe the use of synchrotron X-ray absorption spectroscopy (XAS) and X-ray diffraction (XRD) techniques to probe details of intercalation/deintercalation processes in electrode materials for Li-ion and Na-ion batteries. Both in situ and ex situ experiments are used to understand structural behavior relevant to the operation of devices

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Chemistry

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals
Karthish Manthiram *1,4, Brandon J. Beberwyck *2,4, Dmitri V. Talapin 5,6, A. Paul Alivisatos 2,3,4
1Department of Chemical Engineering, UC Berkeley, 2Department of Materials Science and Engineering, UC Berkeley, 3Department of Chemistry, UC Berkeley, 4Materials Sciences Division, Lawrence Berkeley National Laboratory, 5Department of Chemistry, University of Chicago, 6Center for Nanoscale Materials, Argonne National Laboratory

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

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Engineering

Flame Experiments at the Advanced Light Source: New Insights into Soot Formation Processes
Nils Hansen 1, Scott A. Skeen 1, Hope A. Michelsen 1, Kevin R. Wilson 2, Katharina Kohse-Höinghaus 3
1Combustion Research Facility, Sandia National Laboratories, 2Chemical Sciences Division, Advanced Light Source, Lawrence Berkeley National Laboratory, 3Physikalische Chemie I, Universität Bielefeld

Gas sampling from laboratory-scale flames with online analysis of all species by mass spectrometry is a powerful method to investigate the complex mixture of chemical compounds occurring during combustion processes. Coupled with tunable soft ionization via synchrotron-generated vacuum-ultraviolet radiation, this technique provides isomer-resolved information and potentially fragment-free mass spectra.

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Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements
Prajakta Pradhan Mitra 1,2, Dominique Loqué 1,2
1Feedstocks Division, Joint Bioenergy Institute, 2Physical Biosciences Division, Lawrence Berkeley National Laboratory

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

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Bioengineering

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting
Christopher W. Coyne 1, Karan Patel 1, Johanna Heureaux 1, Jeanne Stachowiak 3, Daniel A. Fletcher 4,5, Allen P. Liu 1,2
1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

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Bioengineering

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Wen-Ting Tsai 1, Ahmed Hassan 1, Purbasha Sarkar 2, Joaquin Correa 1,3, Zoltan Metlagel 1, Danielle M. Jorgens 1, Manfred Auer 1,2
1Life Sciences Division, Lawrence Berkeley National Laboratory, 2Joint Bioenergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 3National Energy Research Scientific Computing Center, Lawrence Berkeley National Laboratory

The bottleneck for cellular 3D electron microscopy is feature extraction (segmentation) in highly complex 3D density maps. We have developed a set of criteria, which provides guidance regarding which segmentation approach (manual, semi-automated, or automated) is best suited for different data types, thus providing a starting point for effective segmentation.

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Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems
Lara Rajeev 1, Eric G. Luning 1, Aindrila Mukhopadhyay 1
1Physical Biosciences Division, Lawrence Berkeley National Laboratory

This video article describes an in vitro microarray based method to determine the gene targets and binding sites for two component system response regulators.

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Bioengineering

Production and Targeting of Monovalent Quantum Dots
Daeha Seo *1,2,3, Justin Farlow *4,5,6, Kade Southard 1,4,7, Young-wook Jun 1,7, Zev J. Gartner 4,5,6,7
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

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Engineering

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities
Han Sae Jung 1,2, Hsin-Zon Tsai 1, Dillon Wong 1, Chad Germany 1, Salman Kahn 1, Youngkyou Kim 1,3, Andrew S. Aikawa 1, Dhruv K. Desai 1, Griffin F. Rodgers 1, Aaron J. Bradley 1, Jairo Velasco Jr. 1, Kenji Watanabe 4, Takashi Taniguchi 4, Feng Wang 1,5,6, Alex Zettl 1,5,6, Michael F. Crommie 1,5,6
1Department of Physics, University of California at Berkeley, 2Department of Chemistry, University of California at Berkeley, 3Department of Chemical and Biomolecular Engineering, University of California at Berkeley, 4National Institute for Materials Science (Japan), 5Materials Sciences Division, Lawrence Berkeley National Laboratory, 6Kavli Energy NanoSciences Institute, University of California at Berkeley and Lawrence Berkeley National Laboratory

This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

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Engineering

Failure Analysis of Batteries Using Synchrotron-based Hard X-ray Microtomography
Katherine J. Harry 1,2, Dilworth Y. Parkinson 3, Nitash P. Balsara 2,4,5
1Department of Materials Science and Engineering, University of California Berkeley, 2Materials Science Division, Lawrence Berkeley National Laboratory, 3Advanced Light Source Division, Lawrence Berkeley National Laboratory, 4Department of Chemical and Biomolecular Engineering, University of California Berkeley, 5Environmental Energy Technology Division, Lawrence Berkeley National Laboratory

Synchrotron-based hard X-ray microtomography is used to image the electrochemical growth of dendrites from a lithium metal electrode through a solid polymer electrolyte membrane.

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Neuroscience

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals
Feng Liu 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

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Engineering

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages
Holly D. Carlton 1, John W. Elmer 1, Yan Li 2, Mario Pacheco 2, Deepak Goyal 2, Dilworth Y. Parkinson 3, Alastair A. MacDowell 3
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2Assembly Test and Technology Development Failure Analysis Labs, Intel Corporation, 3Advanced Light Source, Lawrence Berkeley National Laboratory

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron's high flux and brightness the sample was imaged in just 3 min with an 8.7 µm spatial resolution.

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JoVE Core

Printing Fabrication of Bulk Heterojunction Solar Cells and In Situ Morphology Characterization
Feng Liu 1, Sunzida Ferdous 2, Xianjian Wan 1, Chenhui Zhu 3, Eric Schaible 3, Alexander Hexemer 3, Cheng Wang 3, Thomas P. Russell 1,2
1Materials Sciences Division, Lawrence Berkeley National Laboratory, 2Department of Polymer Science and Engineering, University of Massachusetts, Amherst, 3Advanced Light Source, Lawrence Berkeley National Laboratory

Here, we present a protocol to fabricate organic thin film solar cells using a mini-slot die coater and related in-line structure characterizations using synchrotron scattering techniques.

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Biology

Experimental Approach for Determining Semiconductor/liquid Junction Energetics by Operando Ambient-Pressure X-ray Photoelectron Spectroscopy
Michael Frankston Lichterman *1, Matthias H Richter *2, Shu Hu *1, Ethan J Crumlin *3, Stephanus Axnanda 3, Marco Favaro 3, Walter Drisdell 3, Zahid Hussein 3, Bruce S Brunschwig 4, Nathan S Lewis 1, Zhi Liu 3, Hans-Joachim Lewerenz 2
1Division of Chemistry and Chemical Engineering, California Institute of Technology, 2Joint Center for Artificial Photosynthesis, California Institute of Technology, 3Advanced Light Source, Lawrence Berkeley National Laboratory, 4Beckman Institute, California Institute of Technology

We present a method for the determination of the energy relations of semiconductor/liquid junctions, which are the basis for the successful operation of such renewable solar energy converting systems.

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Chemistry

Synthesis of a Water-soluble Metal–Organic Complex Array
Purnandhu Bose 1, Pradip K. Sukul 1, Omar M. Yaghi 2,3,4, Kentaro Tashiro 1
1International Center for Materials Nanoarchitectonics, National Institute for Materials Science, 2Department of Chemistry, University of California–Berkeley, 3Materials Sciences Division, Lawrence Berkeley National Laboratory, 4Kavli Energy NanoSciences Institute at Berkeley, University of California–Berkeley

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

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Chemistry

Low Pressure Vapor-assisted Solution Process for Tunable Band Gap Pinhole-free Methylammonium Lead Halide Perovskite Films
Carolin M. Sutter-Fella *1,2,3, Yanbo Li *1,4, Nicola Cefarin 1,5,6, Aya Buckley 1,7, Quynh Phuong Ngo 8,9, Ali Javey 2,3, Ian D. Sharp 1, Francesca M. Toma 1
1Joint Center for Artificial Photosynthesis, Chemical Sciences Division, Lawrence Berkeley National Laboratory, 2Electrical Engineering and Computer Sciences, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, 5Department of Physics, Graduate School of Nanotechnology, University of Trieste, 6TASC Laboratory, IOM-CNR - Istituto Officina dei Materiali, 7Department of Chemistry, University of California, Berkeley, 8Materials Science and Engineering, University of California, Berkeley, 9Joint Center for Artificial Photosynthesis, Lawrence Berkeley National Laboratory

Here, we present a protocol for the synthesis of CH3NH3I and CH3NH3Br precursors and the subsequent formation of pinhole-free, continuous CH3NH3PbI3-xBrx thin films for the application in high efficiency solar cells and other optoelectronic devices.

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Biochemistry

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE
Venkataramana R. Pidatala *1,2, Amir Mahboubi *1,2, Jenny C. Mortimer 1,2
1Joint BioEnergy Institute, 2Environmental and Systems Biology, BioSciences Division, Lawrence Berkeley National Laboratory

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

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Developmental Biology

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis
Junhua Lv 1,2, Feng Liu 1,2
1State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

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JoVE Core

Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating
Feng Qin 1, Toshiya Ideue 1, Wu Shi 2, Yijin Zhang 3,4, Ryuji Suzuki 1, Masaro Yoshida 5, Yu Saito 1, Yoshihiro Iwasa 1,5
1Quantum-Phase Electronics Center (QPEC) and Department of Applied Physics, The University of Tokyo, 2Materials Sciences Division, Lawrence Berkeley National Laboratory, 3Institute of Scientific and Industrial Research, Osaka University, 4Max Planck Institute for Solid State Research, 5RIKEN Center for Emergent Matter Science (CEMS)

Here, we present a protocol to control the carrier number in solids by using the electrolyte.

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Environment

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions
Jian Gao 1,2, Joelle Sasse 1,2, Kyle M. Lewald 1,2, Kateryna Zhalnina 1,2, Lloyd T. Cornmesser 1,2, Todd A. Duncombe 3, Yasuo Yoshikuni 2, John P. Vogel 2, Mary K. Firestone 4, Trent R. Northen 1,2
1Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 2Joint Genome Institute, Department of Energy, 3Joint BioEnergy Institute, 4Department of Environmental Science Policy and Management, University of California

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

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Chemistry

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering
Jinpeng Wu 1,2, Shawn Sallis 2,3, Ruimin Qiao 2, Qinghao Li 2,4, Zengqing Zhuo 2,5, Kehua Dai 2,6, Zixuan Guo 2,7, Wanli Yang 2
1Geballe Laboratory for Advanced Materials, Stanford University, 2Advanced Light Source, Lawrence Berkeley National Laboratory, 3Department of Materials Science and Engineering, Binghamton University, 4School of Physics, National Key Laboratory of Crystal Materials, Shandong University, 5School of Advanced Materials, Peking University Shenzhen Graduate School, 6School of Metallurgy, Northeastern University, 7Department of Chemical Engineering, University of California-Santa Barbara

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

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Chemistry

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching
Matthew R. Hauwiller 1, Justin C. Ondry 1, A. Paul Alivisatos 1,2,3,4
1Department of Chemistry, University of California-Berkeley, 2Department of Material Science and Engineering, University of California-Berkeley, 3Kavli Energy NanoScience Institute, 4Materials Sciences Division, Lawrence Berkeley National Laboratory

Graphene liquid cell electron microscopy can be used to observe nanocrystal dynamics in a liquid environment with greater spatial resolution than other liquid cell electron microscopy techniques. Etching premade nanocrystals and following their shape using graphene liquid cell Transmission Electron Microscopy can yield important mechanistic information about nanoparticle transformations.

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JoVE Journal

Synchrotron X-ray Microdiffraction and Fluorescence Imaging of Mineral and Rock Samples
Camelia V. Stan 1, Nobumichi Tamura 1
1Advanced Light Source, Lawrence Berkeley National Laboratory

We describe a beamline setup meant to carry out rapid two-dimensional x-ray fluorescence and x-ray microdiffraction mapping of single crystal or powder samples using either Laue (polychromatic radiation) or powder (monochromatic radiation) diffraction. The resulting maps give information about strain, orientation, phase distribution, and plastic deformation.

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Medicine

Real-Time Dynamic Navigation System for the Precise Quad-Zygomatic Implant Placement in a Patient with a Severely Atrophic Maxilla
Yihan Shen *1, Qinggang Dai *1, Baoxin Tao *1, Wei Huang 2, Feng Wang 2, Kengliang Lan 1, Yuanyuan Sun 1, Xiaowan Ling 1, Lijun Yan 1, Yueping Wang 1, Yiqun Wu 1
1The 2nd Dental Center, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, 2Department of Dental Implantology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology

Here, we present a protocol to achieve precise quad-zygomatic implant placement in patients with severely atrophic maxilla using a real-time dynamic navigation system.

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Environment

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution
Giovanni Gallo 1,2, Martina Aulitto 1,3, Patrizia Contursi 1,4, Danila Limauro 1,4, Simonetta Bartolucci 1, Gabriella Fiorentino 1,4
1Department of Biology, University of Naples Federico II, 2Institute of Polymers, Composites and Biomaterials (IPCB), Consiglio Nazionale delle Ricerche CNR, 3Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, 4BAT Center-Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology, University of Napoli Federico II

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

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Biochemistry

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation
Trinity Cookis 1, Paul Sauer 2,3, Simon Poepsel 4,5, Bong-Gyoon Han 6, Dominik A. Herbst 1,2,6, Robert Glaeser 6, Eva Nogales 1,2,3,6
1Department of Molecular and Cell Biology, University of California, Berkeley, 2California Institute for Quantitative Biology (QB3), University of California, Berkeley, 3Howard Hughes Medical Institute, University of California, Berkeley, 4Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital, University of Cologne, 5Cologne Excellence Cluster for Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 6Molecular Biophysics and Integrative Bio-Imaging Division, Lawrence Berkeley National Laboratory

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.

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