Table Of ContentMethods in
Molecular Biology 1763
Masaru Ishii Editor
Intravital Imaging
of Dynamic Bone
and Immune
Systems
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Intravital Imaging of Dynamic
Bone and Immune Systems
Methods and Protocols
Edited by
Masaru Ishii
Department of Immunology and Cell Biology, Graduate School of Medicine,
Osaka University, Osaka, Osaka, Japan
Editor
Masaru Ishii
Department of Immunology and Cell Biology
Graduate School of Medicine, Osaka University
Osaka, Osaka, Japan
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7761-1 ISBN 978-1-4939-7762-8 (eBook)
https://doi.org/10.1007/978-1-4939-7762-8
Library of Congress Control Number: 2018933038
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Preface
During the last decade, advanced intravital imaging technology by using multiphoton exci-
tation fluorescent microscopy has revolutionized the field of biological sciences. Based on
these techniques, we are now able to visualize in situ behavior of a diversity of living cells
“intravitally” within intact tissues and organs. This research trend would be quite meritori-
ous especially for analyzing bone and immune systems, where various kinds of cell types are
moving around and their spatiotemporal control is pivotal for proper functions in vivo. For
example, bone is a mysterious organ where various kinds of hematopoietic and immune
cells are produced and functioning although poorly analyzed by conventional methodology
such as histological analyses with decalcified bones. Intravital imaging of bones has identi-
fied the behavior of bone cells such as osteoclasts, specialized macrophages contributing to
bone destruction, revealing novel mechanisms controlling their migration and function in
situ. Furthermore, visualization of the dynamic movement of various cell types in lymph
nodes, skin, kidney, nervous systems, and cancer tissues has identified crucial mechanisms
and factors triggering their migration and infiltration in these areas.
Despite the increased importance of the methods in biological sciences and the avail-
ability of expensive imaging modalities such as multiphoton microscopy in many research
institutes, one of the big hurdles preventing popularization of this research trend has so far
been the complicated experimental protocol. Researchers need the procedures to be elabo-
rated for themselves in their respective laboratories.
In this book, leading researchers who are actually doing imaging studies in the field of
bone and immune systems contributed the chapters where they described respective actual
cutting-edge protocols, including some “secret recipes.” These detailed methods would surely
be useful for general readers in order to establish and perform these experiments on their own.
I express my sincere gratitude to all the authors for their willingness to share their secrets
and to Prof. John Walker at Humana Press for giving me the opportunity to publish this book
for the series. Both he and the authors have been patient during the editing of this volume.
Osaka, Japan Masaru Ishii
v
Contents
Preface � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � v
Contributors � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ix
1 Bone Imaging: Osteoclast and Osteoblast Dynamics ........................................... 1
Junichi Kikuta and Masaru Ishii
2 Intravital Imaging of Mouse Bone Marrow:
Hemodynamics and Vascular Permeability .......................................................... 11
Yookyung Jung, Joel A. Spencer, Anthony P. Raphael, Juwell W. Wu,
Clemens Alt, Judith R. Runnels, Briaira Geiger, and Charles P. Lin
3 Bone Imaging: Platelet Formation Dynamics ...................................................... 23
Asuka Sakata and Satoshi Nishimura
4 Live Imaging of Interstitial T Cell Migration
Using Lymph Node Slices .................................................................................. 29
Tomoya Katakai
5 Two-Photon Imaging of T-Cell Motility in Lymph Nodes:
In Vivo and Ex Vivo Approaches ........................................................................ 43
Akira Takeda, Masayuki Miyasaka, and Eiji Umemoto
6 Imaging the Lymph Node Stroma ..................................................................... 53
Clément Ghigo, Rebecca Gentek, and Marc Bajénoff
7 Intravital Imaging of B Cell Responses in Lymph Nodes .................................... 63
Stefano Sammicheli, Mirela Kuka, and Matteo Iannacone
8 Live Imaging of the Skin Immune Responses:
Visualization of the Contact Hypersensitivity Response ...................................... 75
Gyohei Egawa, Tetsuya Honda, and Kenji Kabashima
9 Imaging of Inflammatory Responses in the Mouse Ear Skin ............................... 87
Jackson LiangYao Li, Chi Ching Goh, and Lai Guan Ng
10 In Vivo Imaging of Immune Cells in Peyer’s Patches .......................................... 109
Andrea Reboldi
11 Intravital Imaging of T Cells Within the Spinal Cord .......................................... 119
Naoto Kawakami
12 Kidney Imaging: Intravital Microscopy ............................................................... 129
Takashi Hato, Seth Winfree, and Pierre C. Dagher
13 Intravital Imaging of Liver Cell Dynamics .......................................................... 137
Sayaka Matsumoto, Junichi Kikuta, and Masaru Ishii
14 Intravital Imaging of the Heart at the Cellular Level
Using Two-Photon Microscopy ......................................................................... 145
Ryohei Matsuura, Shigeru Miyagawa, Junichi Kikuta, Masaru Ishii,
and Yoshiki Sawa
vii
viii Contents
15 Imaging Window Device for Subcutaneous Implantation Tumor ........................ 153
Wataru Ikeda, Ken Sasai, and Tsuyoshi Akagi
16 New Tools for Imaging of Immune Systems: Visualization
of Cell Cycle, Cell Death, and Cell Movement by Using
the Mice Lines Expressing Fucci, SCAT3.1, and Kaede and KikGR .................... 165
Michio Tomura
Index ��������������������������������������������������������������������������������������������������������������������������������� 175
Contributors
Tsuyoshi AkAgi • KAN Research Institute Inc�, Kobe, Hyogo, Japan
Clemens AlT • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
mArC BAjénoff • Aix-Marseille University, Centre National de la Recherche Scientifique
(CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre
d’Immunologie de Marseille-Luminy (CIML), Marseille, France
Pierre C. DAgher • Division of Nephrology, Department of Medicine,
Indiana University, Indianapolis, IN, USA
gyohei egAwA • Department of Dermatology, Kyoto University Graduate
School of Medicine, Kyoto, Japan
BriAirA geiger • Department of Chemistry, Richard Stockton College of New Jersey,
Galloway, NJ, USA
reBeCCA genTek • Aix-Marseille University, Centre National de la Recherche Scientifique
(CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre
d’Immunologie de Marseille-Luminy (CIML), Marseille, France
ClémenT ghigo • Aix-Marseille University, Centre National de la Recherche Scientifique
(CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre
d’Immunologie de Marseille-Luminy (CIML), Marseille, France
Chi Ching goh • Singapore Immunology Network (SIgN), A*STAR (Agency for Science,
Technology and Research), Singapore, Singapore; Department of Microbiology,
Immunology Programme, Yong Loo Lin School of Medicine, National University of
Singapore, Singapore, Singapore
TAkAshi hATo • Department of Medicine, Indiana University, Indianapolis, IN, USA
TeTsuyA honDA • Department of Dermatology, Kyoto University Graduate School of
Medicine, Kyoto, Japan
mATTeo iAnnACone • Division of Immunology, Transplantation and Infectious Diseases,
IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele
University, Milan, Italy; Experimental Imaging Center, IRCCS San Raffaele Scientific
Institute, Milan, Italy
wATAru ikeDA • KAN Research Institute Inc�, Kobe, Hyogo, Japan
mAsAru ishii • Department of Immunology and Cell Biology, Graduate School of
Medicine, Osaka University, Osaka, Japan
yookyung jung • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Center
for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul,
Republic of Korea
kenji kABAshimA • Department of Dermatology, Kyoto University Graduate School of
Medicine, Kyoto, Japan
TomoyA kATAkAi • Department of Immunology, Graduate School of Medical and Dental
Sciences, Niigata University, Niigata, Japan
ix
x Contributors
nAoTo kAwAkAmi • Institute of Clinical Neuroimmunology, University Hospital and
Biomedical Center, Ludwig-Maximilians University Munich, Munich, Germany;
Max-Planck Institute of Neurobiology, Martinsried, Germany
juniChi kikuTA • Department of Immunology and Cell Biology, Graduate School of
Medicine, Osaka University, Osaka, Japan
mirelA kukA • Division of Immunology, Transplantation and Infectious Diseases, IRCCS
San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University,
Milan, Italy
jACkson liAngyAo li • Singapore Immunology Network (SIgN), A*STAR (Agency for
Science, Technology and Research), Singapore, Singapore; School of Biological Sciences,
Nanyang Technological University, Singapore, Singapore; CNIC (Fundación Centro
Nacional de Investigaciones Cardiovasculares), Madrid, Spain
ChArles P. lin • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
sAyAkA mATsumoTo • Department of Immunology and Cell Biology, Graduate School of
Medicine, Osaka University, Osaka, Japan
ryohei mATsuurA • Department of Cardiovascular Surgery, Osaka University, Graduate
School of Medicine, Osaka, Japan
shigeru miyAgAwA • Department of Cardiovascular Surgery, Osaka University Graduate
School of Medicine, Osaka, Japan
mAsAyuki miyAsAkA • MediCity Research Laboratory, University of Turku, Turku,
Finland; Interdisciplinary Program for Biomedical Sciences, Institute of Academic
Initiatives, Osaka University, Suita, Osaka, Japan
lAi guAn ng • Singapore Immunology Network (SIgN), A*STAR (Agency for Science,
Technology and Research), Singapore, Singapore; School of Biological Sciences, Nanyang
Technological University, Singapore, Singapore; Department of Microbiology, Immunology
Programme, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore, Singapore
sAToshi nishimurA • Research Division of Cell and Molecular Medicine, Center for
Molecular Medicine, Jichi Medical University, Tochigi, Japan
AnThony P. rAPhAel • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA;
Dermatology Research Centre, Translational Research Institute, School of Medicine, The
University of Queensland, St Lucia, QLD, Australia
AnDreA reBolDi • Department of Pathology, University of Massachusetts Medical School,
Worcester, MA, USA
juDiTh r. runnels • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
AsukA sAkATA • Research Division of Cell and Molecular Medicine, Center for Molecular
Medicine, Jichi Medical University, Tochigi, Japan
sTefAno sAmmiCheli • Division of Immunology, Transplantation and Infectious Diseases,
IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele
University, Milan, Italy
ken sAsAi • KAN Research Institute Inc�, Kobe, Hyogo, Japan
yoshiki sAwA • Department of Cardiovascular Surgery, Osaka University Graduate School
of Medicine, Osaka, Japan
Contributors xi
joel A. sPenCer • Center for Systems Biology and Wellman Center for Photomedicine,
Center for Regenerative Medicine, Massachusetts General Hospital and Harvard Medical
School, Boston, MA, USA; School of Engineering, University of California Merced,
Merced, CA, USA
AkirA TAkeDA • MediCity Research Laboratory, University of Turku, Turku, Finland
miChio TomurA • Laboratory of Immunology, Faculty of Pharmacy, Osaka Ohtani
University, Osaka, Japan
eiji umemoTo • Laboratory of Immune Regulation, Department of Microbiology and
Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan
seTh winfree • Department of Medicine, Indiana University, Indianapolis, IN, USA
juwell w. wu • Center for Systems Biology and Wellman Center for Photomedicine,
Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
