ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of HertfordshireHatfield, Hertfordshire, UK

For further volumes:http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols andmethodologies in the critically acclaimedMethods in Molecular Biology series. The series wasthe first to introduce the step-by-step protocols approach that has become the standard in allbiomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagentsneeded to complete the experiment, and followed by a detailed procedure that is supportedwith a helpful notes section offering tips and tricks of the trade as well as troubleshootingadvice. These hallmark features were introduced by series editor Dr. John Walker andconstitute the key ingredient in each and every volume of the Methods in Molecular Biologyseries. Tested and trusted, comprehensive and reliable, all protocols from the series areindexed in PubMed.

Multiplexed Imaging

Methods and Protocols

Edited by

Eli Zamir

Department of Cellular Biophysics, Max Planck Institute for Medical Research, Stuttgart,Baden-Württemberg, Germany

EditorEli ZamirDepartment of Cellular BiophysicsMax Planck Institute for Medical ResearchStuttgart, Baden-Wurttemberg, Germany

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-0716-1592-8 ISBN 978-1-0716-1593-5 (eBook)https://doi.org/10.1007/978-1-0716-1593-5

© Springer Science+Business Media, LLC, part of Springer Nature 2021, Corrected Publication 2021This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,expressed or implied, with respect to the material contained herein or for any errors or omissions that may have beenmade. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Illustration: Composite scheme illustrating multiplexed imaging of PKA activity, ERK activity, Ca2+ level andcAMP level in living cells. Contributed by Jeremiah Keyes, Sohum Mehta and Jin Zhang. See Chapter 1 for moreinformation.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of SpringerNature.The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface

Multiplexed imaging microscopy consists of methods that enable co-imaging of multiple,different, components in a specimen. Such components can be, for example, the local levelsof distinct proteins, their states (e.g., post-translational modifications), and their interac-tions within cells and tissues. Additionally, multiplexed imaging approaches can include theimaging of biophysical parameters (e.g., biomechanical forces) and combining them withthe imaging of molecular components.

In the era of systems biology, multiplexed imaging increasingly becomes an essentialstrategy for advancing the study of cells and tissues. The main reason for this is thatbiological properties arise from the collective action of multiple distinct biochemical com-ponents affecting each other. Furthermore, these biochemical networks are often organizedin spatiotemporal patterns which are fundamental for the emergence of their biologicalfunctions. Due to intracellular and intercellular variabilities, it is usually infeasible to inferthe spatiotemporal relations between different components simply by imaging separatelyeach of them in different specimens. Multiplexed imaging provides the ultimate solution forthis fundamental challenge, through the co-imaging of multiple components of interest inthe same specimen.

Multiplexed imaging of biochemical networks in intact cells and tissues is fundamentallychallenging. The main challenges here are how to label specifically each of the multiplecomponents of interest in a given specimen and how to image specifically and efficiently thesignal of each of the labels. Fortunately, the frontiers of multiplexed imaging are constantlyadvancing thanks to rapid developments in labeling and imaging approaches. These devel-opments synergize with the rise of systems biology and personalized medicine, whichmotivate multiplexed imaging for basic research and clinical diagnostics.

This volume of the Methods in Molecular Biology series provides a collection of state-of-the-art approaches covering key aspects of multiplexed imaging. Hence, this book shouldbe helpful for researchers interested in implementing multiplexed imaging or in developingnew, cutting-edge multiplexed labeling and imaging techniques.

I am grateful to all the authors for their valuable contributions, providing detailedprotocols and insightful descriptions of their methods. I also would like to thank Dr. JohnM. Walker for his helpful advice along the way. I wish the readers a rewarding use of thisbook and successful multiplexed imaging.

Stuttgart, Baden-Wurttemberg, Germany Eli Zamir

v

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Strategies for Multiplexed Biosensor Imaging to Study IntracellularSignaling Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Jeremiah Keyes, Sohum Mehta, and Jin Zhang

2 Six-Color Confocal Immunofluorescence Microscopy with 4-LaserLines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Lukas Heger, Jennifer J. Luhr, Lukas Amon, Ana-Suncana Smith,Nathalie Eissing, and Diana Dudziak

3 Multiplexed Imaging of Posttranslational Modificationsof Endogenous Proteins in Live Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Yuko Sato and Hiroshi Kimura

4 Multiplex Imaging of Rho GTPase Activities in Living Cells. . . . . . . . . . . . . . . . . . 43Ravi M. Bhalla, Maren Hulsemann, Polina V. Verkhusha,Myla G. Walker, Daria M. Shcherbakova, and Louis Hodgson

5 Multicolor Localization-Based Super Resolution Microscopy . . . . . . . . . . . . . . . . . 69Leila Nahidiazar and Rolf Harkes

6 Multiplexed Tissue Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Evan H. Phillips, David Scholten, Amy C. Flor,Stephen J. Kron, and Steve Seung-Young Lee

7 Multicolor 3D Confocal Imaging of Thick Tissue Sections . . . . . . . . . . . . . . . . . . . 95Leo Kunz and Daniel L. Coutu

8 Multiphoton Deep-Tissue Imaging of Micrometastases andDisseminated Cancer Cells Using Conjugates of Quantum Dotsand Single-Domain Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105Alyona Sukhanova, Fernanda Ramos-Gomes, Patrick Chames,Pavel Sokolov, Daniel Baty, Frauke Alves, and Igor Nabiev

9 Multiplexed Imaging for Immune Profiling on Human FFPE Material . . . . . . . . 125Artur Mezheyeuski and Carina Strell

10 Method for Multiplexed Dynamic Intravital Multiphoton Imaging . . . . . . . . . . . 145Asylkhan Rakhymzhan, Andreas Acs, Ruth Leben, Thomas H. Winkler,Anja E. Hauser, and Raluca A. Niesner

11 Fourier Multiplexed Fluorescence Lifetime Imaging . . . . . . . . . . . . . . . . . . . . . . . . 157Leilei Peng

12 Bimolecular Fluorescence Complementation (BiFC) and MultiplexedImaging of Protein–Protein Interactions in Human Living Cells. . . . . . . . . . . . . . 173Yunlong Jia, Francoise Bleicher, Jonathan Reboulet,and Samir Merabet

vii

13 Out-of-Phase Imaging after Optical Modulation (OPIOM)for Multiplexed Fluorescence Imaging under Adverse OpticalConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191Raja Chouket, Ruikang Zhang, Agnes Pellissier-Tanon,Annie Lemarchand, Agathe Espagne, Thomas Le Saux,and Ludovic Jullien

14 Multicolor Bioluminescence Imaging of Subcellular Structuresand Multicolor Calcium Imaging in Single Living Cells. . . . . . . . . . . . . . . . . . . . . . 229Kazushi Suzuki, Md Nadim Hossain, Tomoki Matsuda,and Takeharu Nagai

15 Nanoparticles for In Vivo Lifetime Multiplexed Imaging . . . . . . . . . . . . . . . . . . . . 239Erving Ximendes, Emma Martın Rodrıguez, Dirk H. Ortgies,Meiling Tan, Guanying Chen, and Blanca del Rosal

16 Versatile on-Demand Fluorescent Labeling of Fusion ProteinsUsing Fluorescence-Activating and Absorption-Shifting Tag (FAST) . . . . . . . . . . 253Arnaud Gautier, Ludovic Jullien, Chenge Li, Marie-Aude Plamont,Alison G. Tebo, Marion Thauvin, Michel Volovitch, and Sophie Vriz

17 UltraPlex Hapten-Based Multiplexed FluorescentImmunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Matt Levin, Amy C. Flor, Helen Snyder, Stephen J. Kron,and David Schwartz

18 Multimodal Approach for Cancer Cell Investigation . . . . . . . . . . . . . . . . . . . . . . . . 289Alexandre Berquand and Jerome Devy

19 Multiplexed Fourier Transform Infrared and Raman Imaging . . . . . . . . . . . . . . . . 299Guillermo Quintas, Bayden R. Wood, Hugh J. Byrne,and David Perez-Guaita

20 Multiplexed Imaging Mass Spectrometry of Histological Staining,N-Glycan and Extracellular Matrix from One Tissue Section:A Tool for Fibrosis Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313Cassandra L. Clift, Anand Mehta, Richard R. Drake,and Peggi M. Angel

21 Multiplexed Raman Imaging in Tissues and Living Organisms . . . . . . . . . . . . . . . 331Travis M. Shaffer and Sanjiv S. Gambhir

Correction to: Out-of-Phase Imaging after Optical Modulation (OPIOM)for Multiplexed Fluorescence Imaging under Adverse OpticalConditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C1

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

viii Contents

Contributors

ANDREAS ACS • Division of Genetics, Department of Biology, Nikolaus-Fiebiger-Center forMolecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen,Germany

FRAUKE ALVES • Max Planck Institute for Experimental Medicine & University MedicalCenter, Gottingen, Germany

LUKAS AMON • Laboratory of Dendritic Cell Biology, Department of Dermatology, UniversityHospital Erlangen, Friedrich-Alexander-University Erlangen-Nurnberg, Erlangen,Germany

PEGGI M. ANGEL • Department of Cell and Molecular Pharmacology and ExperimentalTherapeutics, Medical University of South Carolina, Charleston, SC, USA

DANIEL BATY • Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM,Marseille, France

ALEXANDRE BERQUAND • Laboratoire de Recherche en Nanosciences LRN EA4682 andNanoMat’ platform, Universite de Reims Champagne-Ardenne, Reims, France

RAVI M. BHALLA • Department of Anatomy and Structural Biology, Albert Einstein Collegeof Medicine, Bronx, NY, USA

FRANCOISE BLEICHER • Institut de Genomique Fonctionnelle de Lyon, UMR5242, UniversiteLyon 1, CNRS, Ecole Normale Superieure de Lyon, Lyon Cedex 07, France

HUGH J. BYRNE • FOCAS Research Institute, Technological University Dublin, Dublin,Ireland

PATRICK CHAMES • Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM,Marseille, France

GUANYING CHEN • School of Chemistry and Chemical Engineering, Harbin Institute ofTechnology, Harbin, China

RAJA CHOUKET • PASTEUR, Departement de Chimie, Ecole normale superieure, PSLUniversity, Sorbonne Universite, CNRS, Paris, France

CASSANDRA L. CLIFT • Department of Cell and Molecular Pharmacology and ExperimentalTherapeutics, Medical University of South Carolina, Charleston, SC, USA

DANIEL L. COUTU • Department of Cellular and Molecular Medicine, University of Ottawa,Ottawa, ON, Canada; Regenerative Medicine Program, Ottawa Hospital ResearchInstitute, Ottawa, ON, Canada; Division of Orthopaedic Surgery, The Ottawa Hospital,Ottawa, ON, Canada

BLANCA DEL ROSAL • ARC Centre of Excellence for Nanoscale Biophotonics, RMITUniversity, Melbourne, VIC, Australia

JEROME DEVY • CNRS UMR 7369, Matrice Extracellulaire et Dynamique Cellulaire(MEDyC), UFR Sciences Exactes et Naturelles, Universite de Reims Champagne-Ardenne(URCA), Laboratoire SiRMa - Campus Moulin de la Housse, Reims Cedex, France

RICHARD R. DRAKE • Department of Cell and Molecular Pharmacology and ExperimentalTherapeutics, Medical University of South Carolina, Charleston, SC, USA

DIANA DUDZIAK • Laboratory of Dendritic Cell Biology, Department of Dermatology,University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nurnberg,Erlangen, Germany; Deutsches Zentrum Immuntherapie (DZI), Erlangen, Germany;Comprehensive Cancer Center Erlangen-European Metropolitan Area of Nuremberg

ix

(CCC ER-EMN), Erlangen, Germany; Medical Immunology Campus Erlangen,Erlangen, Germany

NATHALIE EISSING • Laboratory of Dendritic Cell Biology, Department of Dermatology,University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nurnberg,Erlangen, Germany

AGATHE ESPAGNE • PASTEUR, Departement de Chimie, Ecole normale superieure, PSLUniversity, Sorbonne Universite, CNRS, Paris, France

AMY C. FLOR • Department of Molecular Genetics and Cell Biology, The University ofChicago, Chicago, IL, USA

SANJIV S. GAMBHIR • Department of Radiology, Stanford University, Stanford, CA, USA;Department of Bioengineering, Stanford University, Stanford, CA, USA; Bio-X Program,Molecular Imaging Program at Stanford (MIPS), Canary Center for Early CancerDetection, Stanford, CA, USA

ARNAUD GAUTIER • Sorbonne Universite, Ecole Normale Superieure, Universite PSL, CNRS,Laboratoire des Biomolecules, LBM, Paris, France; Institut Universitaire de France, Paris,France

ROLF HARKES • Netherlands Cancer Institute, Amsterdam, NetherlandsANJA E. HAUSER • Immundynamics, Deutsches Rheumaforschungszentrum - a Leibniz

Institute, Berlin, Germany; Immundynamics and Intravital Microscopy, Department ofRheumatology and Clinical Immunology, Charite - Universit€atsmedizin Berlin, CorporateMember of Freie Universit€at Berlin, Humboldt-Universit€at zu Berlin, and Berlin Instituteof Health, Berlin, Germany

LUKAS HEGER • Laboratory of Dendritic Cell Biology, Department of Dermatology,University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nurnberg,Erlangen, Germany

LOUIS HODGSON • Department of Anatomy and Structural Biology, Albert Einstein Collegeof Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College ofMedicine, Bronx, NY, USA

MD NADIM HOSSAIN • SANKEN (The Institute of Scientific and Industrial Research),Osaka University, Osaka, Ibaraki, Japan

MAREN HULSEMANN • Department of Anatomy and Structural Biology, Albert EinsteinCollege of Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert EinsteinCollege of Medicine, Bronx, NY, USA

YUNLONG JIA • Institut de Genomique Fonctionnelle de Lyon, UMR5242, Universite Lyon 1,CNRS, Ecole Normale Superieure de Lyon, Lyon Cedex 07, France

LUDOVIC JULLIEN • PASTEUR, Department of Chemistry, Ecole Normale Superieure,Universite PSL, Sorbonne Universite, CNRS, Paris, France

JEREMIAH KEYES • Department of Pharmacology, University of California, San Diego, LaJolla, CA, USA

HIROSHI KIMURA • Cell Biology Center, Institute of Innovative Research, Tokyo Institute ofTechnology, Tokyo, Japan

STEPHEN J. KRON • Department of Molecular Genetics and Cell Biology, The University ofChicago, Chicago, IL, USA; Ludwig Center for Metastasis Research, The University ofChicago, Chicago, IL, USA

LEO KUNZ • Department of Biosystems Science and Engineering, ETH Zurich, Basel,Switzerland; Roche Innovation Center Zurich, Pharmaceutical Research & EarlyDevelopment (pRED), Schlieren, Switzerland

x Contributors

RUTH LEBEN • Biophysical Analytics, Deutsches Rheumaforschungszentrum - a LeibnizInstitute, Berlin, Germany

STEVE SEUNG-YOUNG LEE • Department of Pharmaceutical Sciences, The University ofIllinois at Chicago, Chicago, IL, USA

ANNIE LEMARCHAND • Laboratoire de Physique Theorique de la Matiere Condensee (LPTMC), Sorbonne Universite, Centre National de la Recherche Scientifique (CNRS),Paris, France

MATT LEVIN • Cell IDx, San Diego, CA, USACHENGE LI • PASTEUR, Department of Chemistry, Ecole Normale Superieure, Universite

PSL, Sorbonne Universite, CNRS, Paris, France; Department of Obstetrics and Gynecology,Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China;State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren JiHospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

JENNIFER J. LUHR • Department of Physics, Nano-Optics, Sandoghdar Division, Max PlanckInstitute for the Science of Light, Erlangen, Germany

EMMA MARTIN RODRIGUEZ • Nanomaterials for BioImaging Group, Instituto Ram�on yCajal de Investigaci�on Sanitaria IRYCIS, Madrid, Spain; Fluorescence Imaging Group,Departamento de Fısica Aplicada, Facultad de Ciencias, Universidad Aut�onoma deMadrid, Madrid, Spain

TOMOKI MATSUDA • SANKEN (The Institute of Scientific and Industrial Research), OsakaUniversity, Osaka, Ibaraki, Japan

ANAND MEHTA • Department of Cell and Molecular Pharmacology and ExperimentalTherapeutics, Medical University of South Carolina, Charleston, SC, USA

SOHUM MEHTA • Department of Pharmacology, University of California, San Diego, LaJolla, CA, USA

SAMIR MERABET • Institut de Genomique Fonctionnelle de Lyon, UMR5242, Universite Lyon1, CNRS, Ecole Normale Superieure de Lyon, Lyon Cedex 07, France

ARTUR MEZHEYEUSKI • Department of Immunology, Genetics and Pathology, UppsalaUniversity, Uppsala, Sweden

IGOR NABIEV • Laboratoire de Recherche en Nanosciences, LRN-EA4682, Universite deReims Champagne-Ardenne, Reims, France; Laboratory of Nano-Bioengineering,National Research Nuclear University MEPhI (Moscow Engineering Physics Institute),Moscow, Russia

TAKEHARU NAGAI • SANKEN (The Institute of Scientific and Industrial Research), OsakaUniversity, Osaka, Ibaraki, Japan

LEILA NAHIDIAZAR • Netherlands Cancer Institute, Amsterdam, NetherlandsRALUCA A. NIESNER • Biophysical Analytics, Deutsches Rheumaforschungszentrum - a

Leibniz Institute, Berlin, Germany; Dynamic and Functional in vivo Imaging, Institutefor Veterinary Physiology, Veterinary Medicine, Freie Universit€at Berlin, Berlin, Germany

DIRK H. ORTGIES • Nanomaterials for BioImaging Group, Instituto Ram�on y Cajal deInvestigaci�on Sanitaria IRYCIS, Madrid, Spain; Nanomaterials for BioImaging Group,Departamento de Fısica de Materiales, Facultad de Ciencias, Universidad Aut�onoma deMadrid, Madrid, Spain

AGNES PELLISSIER-TANON • PASTEUR, Departement de Chimie, Ecole normale superieure,PSL University, Sorbonne Universite, CNRS, Paris, France

LEILEI PENG • Wyant College of Optical Science, University of Arizona, Tucson, AZ, USADAVID PEREZ-GUAITA • FOCAS Research Institute, Technological University Dublin,

Dublin, Ireland

Contributors xi

EVAN H. PHILLIPS • Department of Pharmaceutical Sciences, The University of Illinois atChicago, Chicago, IL, USA

MARIE-AUDE PLAMONT • PASTEUR, Department of Chemistry, Ecole Normale Superieure,Universite PSL, Sorbonne Universite, CNRS, Paris, France

GUILLERMO QUINTAS • Health & Biomedicine, LEITAT Technological Center, Barcelona,Spain

ASYLKHAN RAKHYMZHAN • Biophysical Analytics, Deutsches Rheumaforschungszentrum - aLeibniz Institute, Berlin, Germany

FERNANDA RAMOS-GOMES • Max Planck Institute for Experimental Medicine & UniversityMedical Center, Gottingen, Germany

JONATHAN REBOULET • Institut de Genomique Fonctionnelle de Lyon, UMR5242, UniversiteLyon 1, CNRS, Ecole Normale Superieure de Lyon, Lyon Cedex 07, France

YUKO SATO • Cell Biology Center, Institute of Innovative Research, Tokyo Institute ofTechnology, Tokyo, Japan

THOMAS LE SAUX • PASTEUR, Departement de Chimie, Ecole normale superieure, PSLUniversity, Sorbonne Universite, CNRS, Paris, France

DAVID SCHOLTEN • Department of Molecular Genetics and Cell Biology, The University ofChicago, Chicago, IL, USA

DAVID SCHWARTZ • Cell IDx, San Diego, CA, USATRAVIS M. SHAFFER • Department of Radiology, Stanford University, Stanford, CA, USADARIA M. SHCHERBAKOVA • Department of Anatomy and Structural Biology, Albert Einstein

College of Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert EinsteinCollege of Medicine, Bronx, NY, USA

ANA-SUNCANA SMITH • Physics Underlying Life Sciences Group, Institute for TheoreticalPhysics, Friedrich-Alexander-University Erlangen-Nurnberg, Erlangen, Germany; Groupfor Computational Life Sciences, Division of Physical Chemistry, Institute Ruaer Boskovic,Zagreb, Croatia

HELEN SNYDER • Cell IDx, San Diego, CA, USAPAVEL SOKOLOV • Laboratory of Nano-Bioengineering, National Research Nuclear

University MEPhI (Moscow Engineering Physics Institute), Moscow, RussiaCARINA STRELL • Department of Immunology, Genetics and Pathology, Uppsala University,

Uppsala, SwedenALYONA SUKHANOVA • Laboratoire de Recherche en Nanosciences, LRN-EA4682, Universite

de Reims Champagne-Ardenne, Reims, FranceKAZUSHI SUZUKI • Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku,

Tokyo, Japan; SANKEN (The Institute of Scientific and Industrial Research), OsakaUniversity, Osaka, Ibaraki, Japan

MEILING TAN • School of Chemistry and Chemical Engineering, Harbin Institute ofTechnology, Harbin, China

ALISON G. TEBO • Sorbonne Universite, Ecole Normale Superieure, Universite PSL, CNRS,Laboratoire des Biomolecules, LBM, Paris, France; Janelia Farms Research Campus,Howard Hughes Medical Institute, Ashburn, VA, USA

MARION THAUVIN • Center for Interdisciplinary Research in Biology (CIRB), College deFrance, CNRS, INSERM, Universite PSL, Paris, France

POLINA V. VERKHUSHA • Department of Anatomy and Structural Biology, Albert EinsteinCollege of Medicine, Bronx, NY, USA

xii Contributors

MICHEL VOLOVITCH • Center for Interdisciplinary Research in Biology (CIRB), College deFrance, CNRS, INSERM, Universite PSL, Paris, France; Department of Biology, EcoleNormale Superieure, Universite PSL, Paris, France

SOPHIE VRIZ • Center for Interdisciplinary Research in Biology (CIRB), College de France,CNRS, INSERM, Universite PSL, Paris, France; Faculty of Science, Universite de Paris,Paris, France

MYLA G. WALKER • Department of Anatomy and Structural Biology, Albert Einstein Collegeof Medicine, Bronx, NY, USA

THOMAS H. WINKLER • Division of Genetics, Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg,Erlangen, Germany

BAYDEN R. WOOD • Faculty of Science, Centre for Biospectroscopy, School of Chemistry,Monash University, Clayton, VIC, Australia

ERVING XIMENDES • Nanomaterials for BioImaging Group, Instituto Ram�on y Cajal deInvestigaci�on Sanitaria IRYCIS, Madrid, Spain; Nanomaterials for BioImaging Group,Departamento de Fısica de Materiales, Facultad de Ciencias, Universidad Aut�onoma deMadrid, Madrid, Spain

JIN ZHANG • Department of Pharmacology, University of California, San Diego, La Jolla,CA, USA

RUIKANG ZHANG • PASTEUR, Departement de Chimie, Ecole normale superieure, PSLUniversity, Sorbonne Universite, CNRS, Paris, France

Contributors xiii