Nagoya University View Institution's Website 11 articles published in JoVE Immunology and Infection Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana Rikako Hirata*1, Momoko Takagi*2, Yosuke Toda2,3, Akira Mine1 1Graduate School of Agriculture, Kyoto University, 2Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, 3Phytometrics Co., Ltd. Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device. Behavior Tracking Sugar-Elicited Local Searching Behavior in Drosophila Manal Shakeel1,2, Shivam Kaushik3, Teiichi Tanimura4, Axel Brockmann1, Pinky Kain3,5 1National Centre for Biological Sciences, 2The University of Trans-Disciplinary Health Sciences and Technology, 3Regional Centre for Biotechnology, 4Neural Circuit Group, Division of Biological Science, Graduate School of Science, Nagoya University, 5Perelman School of Medicine, University of Pennsylvania This protocol describes a behavioral assay for recording sugar-elicited search behavior using Drosophila melanogaster. The assay can be utilized to study feeding and foraging-related behaviors, as well as the underlying neuronal mechanisms. Chemistry Magnetometric Characterization of Intermediates in the Solid-State Electrochemistry of Redox-Active Metal-Organic Frameworks Qi Chen1, Zhongyue Zhang2, Kunio Awaga1,3 1Department of Chemistry, Graduate School of Science, Nagoya University, 2International Research Organization for Advanced Science and Technology, Kumamoto University, 3Integrated Research Consortium on Chemical Sciences, Nagoya University Ex situ magnetic surveys can directly provide bulk and local information on a magnetic electrode to reveal its charge storage mechanism step by step. Herein, electron spin resonance (ESR) and magnetic susceptibility are demonstrated to monitor the evaluation of paramagnetic species and their concentration in a redox-active metal-organic framework (MOF). Biology Deep Fluorescence Observation in Rice Shoots via Clearing Technology Yoko Niimi1, Keisuke Nagai2, Motoyuki Ashikari2, Yoko Mizuta3,4 1Graduate School of Bioagricultural Sciences, Nagoya University, 2Bioscience and Biotechnology Center, Nagoya University, 3Institute for Advanced Research (IAR), Nagoya University, 4Institute of Transformative Bio-Molecules (ITbM), Nagoya University The present protocol describes a clearing technique for rice shoots, which are difficult to prepare for internal structural observations owing to the hard, thick, or layered nature of the tissues. This method facilitates continuous and deep fluorescence observations, even in adult rice plants. Biology Optical Clearing of Plant Tissues for Fluorescence Imaging Daisuke Kurihara1, Yoko Mizuta1,2, Shiori Nagahara1, Yoshikatsu Sato1,3, Tetsuya Higashiyama1,3,4 1Institute of Transformative Bio-Molecules (ITbM), Nagoya University, 2Institute for Advanced Research (IAR), Nagoya University, 3Division of Biological Science, Graduate School of Science, Nagoya University, 4Department of Biological Sciences, Graduate School of Science, The University of Tokyo Here, a method is described for making plant tissues transparent while maintaining the stability of fluorescent proteins. This technique facilitates deep imaging of cleared plant tissues without physical sectioning. Chemistry Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis Masahiro Ohtsuka1, Shunsuke Muto1 1Electron Nanoscopy Division, Advanced Measurement Technology Center, Institute of Materials & Systems for Sustainability, Nagoya University We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects. Neuroscience Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila Hiroshi Ishimoto1, Hiroko Sano2 1Division of Biological Science, Graduate School of Science, Nagoya University, 2Department of Molecular Genetics, Institute of Life Science, Kurume University This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain. Bioengineering Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces Naoki Yanagisawa1, Nagisa Sugimoto2, Tetsuya Higashiyama1,2, Yoshikatsu Sato2 1Division of Biological Science, Graduate School of Science, Nagoya University, 2Institute of Transformative Bio-Molecules (ITbM), Nagoya University We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 µm) in a microfluidic device. Developmental Biology In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana Daisuke Kurihara*1,2, Yusuke Kimata*1, Tetsuya Higashiyama1,2,3, Minako Ueda1,3 1Division of Biological Science, Graduate School of Science, Nagoya University, 2Higashiyama Live-Holonics Project, JST-ERATO, Nagoya University, 3Institute of Transformative Bio-Molecules (ITbM), Nagoya University This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos. Neuroscience Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo Ariel M. Lyons-Warren1, Tsunehiko Kohashi1,2, Steven Mennerick3, Bruce A. Carlson1 1Department of Biology, Washington University in St. Louis, 2Division of Biological Science, Graduate School of Science, Nagoya University, 3Department of Psychiatry, Washington University in St. Louis Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches. Biology Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy R. Craig Everroad*1, Seiji Yoshida*2, Yuuri Tsuboi1, Yasuhiro Date3, Jun Kikuchi2,3,4, Shigeharu Moriya1,2 1Biosphere Oriented Biology Research Unit, RIKEN Advanced Science Institute, 2Graduate School of Nanobioscience, Yokohama City University, 3Advanced NMR Metabomics Research Team, RIKEN Plant Science Center, 4Graduate School of Bioagricultural Science, Nagoya University A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.