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Nagoya University

12 ARTICLES PUBLISHED IN JoVE

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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 Tsuboi 1, Yasuhiro Date 3, Jun Kikuchi 2,3,4, Shigeharu Moriya 1,2
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.

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Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo
Ariel M. Lyons-Warren 1, Tsunehiko Kohashi 1,2, Steven Mennerick 3, Bruce A. Carlson 1
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.

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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 Higashiyama 1,2,3, Minako Ueda 1,3
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.

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Bioengineering

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces
Naoki Yanagisawa 1, Nagisa Sugimoto 2, Tetsuya Higashiyama 1,2, Yoshikatsu Sato 2
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.

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Neuroscience

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila
Hiroshi Ishimoto 1, Hiroko Sano 2
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.

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Chemistry

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis
Masahiro Ohtsuka 1, Shunsuke Muto 1
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.

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JoVE Journal

Optical Clearing of Plant Tissues for Fluorescence Imaging
Daisuke Kurihara 1, Yoko Mizuta 1,2, Shiori Nagahara 1, Yoshikatsu Sato 1,3, Tetsuya Higashiyama 1,3,4
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.

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Biology

Deep Fluorescence Observation in Rice Shoots via Clearing Technology
Yoko Niimi 1, Keisuke Nagai 2, Motoyuki Ashikari 2, Yoko Mizuta 3,4
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.

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Biology

Immunocytochemical Visualization of Proteins from Cyanobacterial Cells with High Autofluorescence of Phycoerythrin and Phycourobilin
Takako Masuda 1,2, Dominika Majerová 1, Kasia Piwosz 1,3, Tatsuhiro Tsurumaki 1, Yuichi Fujita 4, Ondřej Prášil 1
1Institute of Microbiology, The Czech Academy of Sciences, 2Japan Fisheries Research and Education Agency, 3National Marine Fisheries Research Institute, 4Graduate School of Bioagricultural Sciences, Nagoya University

This protocol outlines the visualization and quantification of a particular protein within cells at the cellular level for the phycoerythrin-containing cyanobacterium, Crocosphaera watsonii.

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Chemistry

Magnetometric Characterization of Intermediates in the Solid-State Electrochemistry of Redox-Active Metal-Organic Frameworks
Qi Chen 1, Zhongyue Zhang 2, Kunio Awaga 1,3
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).

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Behavior

Tracking Sugar-Elicited Local Searching Behavior in Drosophila
Manal Shakeel 1,2, Shivam Kaushik 3, Teiichi Tanimura 4, Axel Brockmann 1, Pinky Kain 3,5
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.

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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 Toda 2,3, Akira Mine 1
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.

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