Academia Sinica 27 articles published in JoVE Neuroscience Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish Jose Sandino Bandonil*1,2, Yi-Hsuan Liao*1, Amir Fathi1, Kuo-Hua Huang1,2 1Institute of Molecular Biology, Academia Sinica, 2Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. Biology Monitoring Stub1-Mediated Pexophagy Bo-Hua Chen1, Wei Yuan Yang1,2 1Institute of Biological Chemistry, Academia Sinica, 2Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University This protocol provides instructions for triggering and monitoring Stub1-mediated pexophagy in live cells. Immunology and Infection A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture Yu-Hsien Hung1, Hsiu-Jung Wang1, Jen-Shuang Wang1, Chia-Hao Hsu1, Huan-Yuan Chen1 1Inflammation Core Facility, Institute of Biomedical Sciences, Academia Sinica AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells. Biochemistry Preparation of High-Temperature Sample Grids for Cryo-EM Yuan-Chih Chang1,2, Chin-Yu Chen3,4, Ming-Daw Tsai1,5 1Institute of Biological Chemistry, Academia Sinica, 2Academia Sinica Cryo-EM Facility, Academia Sinica, 3Department of Life Sciences, National Central University, 4Hubei University, 5Institute of Biochemical Sciences, National Taiwan University This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 °C, prior to plunge freezing for cryo-EM experiments. Bioengineering Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices Hui-Fang Chang1, Shih-En Chou1, Ji-Yen Cheng1,2,3,4 1Research Center for Applied Sciences, Academia Sinica, 2Institute of Biophotonics, National Yang Ming Chao Tung University, 3Department of Mechanical and Mechatronic Engineering, National Taiwan Ocean University, 4College of Engineering, Chang Gung University In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system. Chemistry Hierarchical and Programmable One-Pot Oligosaccharide Synthesis Cheng-Wei Cheng1, Yixuan Zhou1, Wen-Harn Pan2, Supriya Dey3, Chung-Yi Wu1, Wen-Lian Hsu4, Chi-Huey Wong1,3 1Genomics Research Center, Academia Sinica, 2Institute of Biomedical Sciences, Academia Sinica, 3Department of Chemistry, Scripps Research Institute, 4Institute of Information Science, Academia Sinica This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination experiments and one-pot glycosylation of SSEA-4. Engineering One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes Chien-Kai Wang1,2, Wei-Hao Liao1, Hsiao-Mei Wu1, Yi-Chung Tung1,3 1Research Center for Applied Sciences, Academia Sinica, 2Department of Civil Engineering, Tamkang University, 3College of Engineering, Chang Gung University Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching. Chemistry An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Yu-Meng Ou1,2, Shu-Yun Kuo1, Hsun Lee1, Huan-Tsung Chang2, Yi-Sheng Wang1 1Genomic Research Center, Academia Sinica, 2Department of Chemistry, National Taiwan University A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated. Neuroscience Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy Ee Shan Liau*1,2, Ya-Ping Yen*2,3, Jun-An Chen1,2 1Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, 2Institute of Molecular Biology, Academia Sinica, 3Institute of Biotechnology, College of Bio-Resources and Agriculture, National Taiwan University Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system. Engineering Well-aligned Vertically Oriented ZnO Nanorod Arrays and their Application in Inverted Small Molecule Solar Cells Ming-Yi Lin1, Shang-Hsuan Wu2, Li-Jen Hsiao3, Widhya Budiawan2, Shih-Lun Chen4, Wei-Chen Tu4, Chia-Yen Lee1, Yia-Chung Chang2, Chih-Wei Chu2 1Department of Electrical Engineering, National United University, 2Research Center of Applied Science, Academia Sinica, 3Department of Graduate Institute of Photonics and Optoelectronics, National Taiwan University, 4Department of Electronic Engineering, Chung Yuan Christian University This manuscript describes how to design and fabricate efficient inverted SMPV1:PC71BM solar cells with ZnO nanorods (NRs) grown on a high quality Al-doped ZnO (AZO) seed layer. The well-aligned vertically oriented ZnO NRs exhibit high crystalline properties. The power conversion efficiency of solar cells can reach 6.01%. Chemistry Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling Sachin S. Shivatare1, Vidya S. Shivatare1, Chung-Yi Wu1, Chi-Huey Wong1 1Genomics Research Center, Academia Sinica A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated. Neuroscience Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms Bo-Jeng Wang*1, Pei-Yi Wu*1, Yun-Wen Chen*1, Yu-Tzu Chang1, Noopur Bhore1, Po-Fan Wu1, Yung-Feng Liao1 1Laboratory of Molecular Neurobiology, Institute of Cellular and Organismic Biology, Academia Sinica We have successfully generated two substrate-specific γ-secretase assays. Both cell-based assays presented here are designed to quantify γ-secretase enzymatic activities via the output of firefly luciferase reporters. Biology 3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography Yi-fan Jiang1, Hsiang-ling Lin2, Chi-yu Fu2 1Graduate Institute of Molecular and Comparative Pathobiology, School of Veterinary Medicine, National Taiwan University, 2Institute of Cellular and Organismic Biology, Academia Sinica In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Engineering Preparation of Large-area Vertical 2D Crystal Hetero-structures Through the Sulfurization of Transition Metal Films for Device Fabrication Chong-Rong Wu1,2, Tung-Wei Chu2,3, Kuan-Chao Chen1,2, Shih-Yen Lin1,2 1Graduate Institute of Electronics Engineering, National Taiwan University, 2Research Center for Applied Sciences, Academia Sinica, 3Graduate Institute of Photonics and Optoelectronics, National Taiwan University Through the sulfurization of pre-deposited transition metals, large-area and vertical 2D crystal hetero-structures can be fabricated. The film transferring and device fabrication procedures are also demonstrated in this report. Bioengineering An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation Hua-De Gao1,2, Pounraj Thanasekaran1, Tzu-Ho Chen1,2, Yu-Hsu Chang3, Yu-Ju Chen1,2, Hsien-Ming Lee1 1Institute of Chemistry, Academia Sinica, 2Department of Chemistry, National Taiwan University, 3Department of Materials and Mineral Resources Engineering, National Taipei University of Technology In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol. Developmental Biology Long-term Live Imaging of Drosophila Eye Disc Chia-Kang Tsao1,2, Hui-Yu Ku1,2, Y. Henry Sun1,2 1Institute of Genomic Sciences, National Yang-Ming University, 2Institute of Molecular Biology, Academia Sinica This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup. Immunology and Infection Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope Chien-Wen Chen1, Chi-Yao Chang1 1Institute of Cellular and Organismic Biology, Academia Sinica Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications. Developmental Biology Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM Hung-Chang Shen1,2, Tsai-Chi Hsu1, Pei-Chi Chung1, Hung-Hsiang Yu1,2 1Institute of Cellular and Organismic Biology, Academia Sinica, 2Graduate Institute of Life Sciences, National Defense Medical Center Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals. Bioengineering Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients Hung-Ju Chiang1, Sih-Ling Yeh1, Chien-Chung Peng1, Wei-Hao Liao1, Yi-Chung Tung1 1Research Center for Applied Sciences, Academia Sinica The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation. Biology Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples Tien-Kuan Chen*1, Hui-Ting Yang*1, Su-Chiung Fang1, Yi-Chen Lien1, Ting-Ting Yang1, Swee-Suak Ko1 1Academia Sinica Biotechnology Center in Southern Taiwan; Agricultural Biotechnology Research Center, Academia Sinica This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH). Engineering Effect of Bending on the Electrical Characteristics of Flexible Organic Single Crystal-based Field-effect Transistors Man-Tzu Ho1,2, Yu-Tai Tao1 1Institution of Chemistry, Academia Sinica, 2Department of Chemistry, National Central University This manuscript describes the bending process of an organic single crystal-based field-effect transistor to maintain a functioning device for electronic property measurement. The results suggest that bending causes changes in the molecular spacing in the crystal and thus in the charge hopping rate, which is important in flexible electronics. Biochemistry Preparation of Homogeneous MALDI Samples for Quantitative Applications Yu-Meng Ou1,2, Chien-Wei Tsao1,3, Yin-Hung Lai1, Hsun Lee1, Huan-Tsung Chang2, Yi-Sheng Wang1 1Genomic Research Center, Academia Sinica, 2Department of Chemistry, National Taiwan University, 3Department of Chemical Engineering and Biotechnology, National Taipei University of Technology A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated. Bioengineering Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli Tzu-Yuan Chou1, Yung-Shin Sun2, Hsien-San Hou3, Shang-Ying Wu1, Yun Zhu1, Ji-Yen Cheng3, Kai-Yin Lo1 1Department of Agricultural Chemistry, National Taiwan University, 2Department of Physics, Fu-Jen Catholic University, 3Research Center for Applied Sciences, Academia Sinica Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli. Behavior Conscious and Non-conscious Representations of Emotional Faces in Asperger's Syndrome Vincent S. C. Chien1,2, Arthur C. Tsai1, Han Hsuan Yang3, Yi-Li Tseng4, Alexander N. Savostyanov5,6, Michelle Liou1,7 1Institute of Statistical Science, Academia Sinica, 2Max Planck Institute for Human Cognitive and Brain Sciences, 3Department of Psychology, Fo Guang University, 4Department of Electrical Engineering, Fu Jen Catholic University, 5State Research Institute of Physiology and Basic Medicine, 6Novosibirsk State University, 7Imaging Research Center, Taipei Medical University An EEG experimental protocol is designed to clarify the interplay between conscious and non-conscious representations of emotional faces in patients with Asperger's syndrome. The technique suggests that patients with Asperger's syndrome have deficits in non-conscious representation of emotional faces, but have comparable performance in conscious representation with healthy controls. Medicine Autoradiographic Measurements of [14C]-Iodoantipyrine in Rat Brain Following Central Post-Stroke Pain Andrew Chih Wei Huang*1, Hsiang-Chin Lu*2, Bai Chuang Shyu2 1Department of Psychology, Fo Guang University, 2Institute of Biomedical Sciences, Academia Sinica We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model. Neuroscience Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation Hsiang-Chin Lu1, Wei-Jen Chang1, Wei-Pang Chang2, Bai-Chuang Shyu1 1Institute of Biomedical Sciences, Academia Sinica, 2Department of Anesthesiology, University of Alabama at Birmingham Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated. Bioengineering Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip Hsien-San Hou1, Hui-Fang Chang1, Ji-Yen Cheng1,2,3,4,5 1Research Center for Applied Sciences, Academia Sinica, 2Institute of Biophotonics, National Yang-Ming University, 3Biophotonics & Molecular Imaging Research Center (BMIRC), National Yang-Ming University, 4Department of Mechanical and Mechatronic Engineering, National Taiwan Ocean University, 5Ph.D. Program in Microbial Genomics, National Chung Hsing University Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.