Explore the full ebook collection and download it now at textbookfull.

com

Intravital Imaging of Dynamic Bone and Immune

Systems Methods and Protocols 1st Edition Masaru
Ishii (Eds.)

https://textbookfull.com/product/intravital-imaging-of-
dynamic-bone-and-immune-systems-methods-and-protocols-1st-
edition-masaru-ishii-eds/

OR CLICK HERE

DOWLOAD EBOOK

Browse and Get More Ebook Downloads Instantly at https://textbookfull.com

Click here to visit textbookfull.com and download textbook now
 Your digital treasures (PDF, ePub, MOBI) await
Download instantly and pick your perfect format...

Read anywhere, anytime, on any device!

Intravital Imaging of Dynamic Bone and Immune Systems

Methods and Protocols 1st Edition Masaru Ishii (Eds.)

https://textbookfull.com/product/intravital-imaging-of-dynamic-bone-
and-immune-systems-methods-and-protocols-1st-edition-masaru-ishii-eds/

textbookfull.com

Suppression and Regulation of Immune Responses Methods and

Protocols Volume II 1st Edition Maria Cristina Cuturi

https://textbookfull.com/product/suppression-and-regulation-of-immune-
responses-methods-and-protocols-volume-ii-1st-edition-maria-cristina-
cuturi/
textbookfull.com

RNA Imaging Methods and Protocols 1st Edition Zdravka

Medarova (Eds.)

https://textbookfull.com/product/rna-imaging-methods-and-
protocols-1st-edition-zdravka-medarova-eds/

textbookfull.com

Systems Metabolic Engineering Methods and Protocols 1st

Edition Stephan Pabinger

https://textbookfull.com/product/systems-metabolic-engineering-
methods-and-protocols-1st-edition-stephan-pabinger/

textbookfull.com
Systems Chemical Biology: Methods and Protocols Slava
Ziegler

https://textbookfull.com/product/systems-chemical-biology-methods-and-
protocols-slava-ziegler/

textbookfull.com

Yeast Systems Biology Methods and Protocols Stephen G.

Oliver

https://textbookfull.com/product/yeast-systems-biology-methods-and-
protocols-stephen-g-oliver/

textbookfull.com

Proteomics in Systems Biology Methods and Protocols 1st

Edition Jörg Reinders (Eds.)

https://textbookfull.com/product/proteomics-in-systems-biology-
methods-and-protocols-1st-edition-jorg-reinders-eds/

textbookfull.com

Cancer Systems Biology Methods and Protocols 1st Edition

Louise Von Stechow (Eds.)

https://textbookfull.com/product/cancer-systems-biology-methods-and-
protocols-1st-edition-louise-von-stechow-eds/

textbookfull.com

Bone Research Protocols Aymen I. Idris

https://textbookfull.com/product/bone-research-protocols-aymen-i-
idris/

textbookfull.com
Methods in
Molecular Biology 1763

Masaru Ishii Editor

Intravital Imaging
of Dynamic Bone
and Immune
Systems
Methods and Protocols
Methods in Molecular Biology

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

© Springer Science+Business Media, LLC 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on 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 regulations
and 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 to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

The registered company is Springer Science+Business Media, LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
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
 Chapter 1

Bone Imaging: Osteoclast and Osteoblast Dynamics

Junichi Kikuta and Masaru Ishii

Abstract
Bone is continually remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts. Although it
has long been believed that bone homeostasis is tightly regulated by communication between osteoclasts
and osteoblasts, the fundamental process and dynamics have remained elusive. To resolve this, we estab-
lished an intravital bone imaging system using multiphoton microscopy to visualize mature osteoclasts and
osteoblasts in living bone.
We herein describe the methodology for visualizing the in vivo behavior of bone-resorbing osteoclasts
and bone-forming osteoblasts in living bone tissues using intravital multiphoton microscopy. This approach
facilitates investigation of cellular dynamics in the pathogenesis of bone-destructive disorders, such as
osteoporosis and rheumatoid arthritis in vivo, and would thus be useful for evaluating the efficacy of novel
anti-bone-resorptive drugs.

Key words Intravital imaging, Multiphoton microscopy, Osteoclast, Osteoblast, pH-sensing probe

1 Introduction

Bone is a dynamic tissue that undergoes continuous remodeling by

bone-resorbing osteoclasts and bone-forming osteoblasts [1].
Tight control of bone remodeling is critical for maintaining bone
homeostasis in response to structural and metabolic demands.
Bone remodeling is strictly controlled through a complex commu-
nication network between osteoblast- and osteoclast-­lineage cells
[2]. Therefore, it is essential to understand the spatial–temporal
relationship and interactions between osteoblasts and osteoclasts
in vivo. In particular, it remains controversial whether these cell
types physically interact with each other.
Bone is the hardest tissue in the body; for this reason, it is tech-
nically difficult to visualize cellular interactions in the bone marrow
cavities of living animals. The morphology and structure of bone
tissues can be analyzed using various conventional methods,
including micro-computed tomography, histomorphological anal-
yses, and flow cytometry. These methods yield information on cell

Masaru Ishii (ed.), Intravital Imaging of Dynamic Bone and Immune Systems: Methods and Protocols, Methods in Molecular Biology,
vol. 1763, https://doi.org/10.1007/978-1-4939-7762-8_1, © Springer Science+Business Media, LLC 2018

1
2 Junichi Kikuta and Masaru Ishii

shape and gene expression patterns, but not on dynamic cell move-
ments in living bone marrow. The recent introduction of fluores-
cence microscopy has enabled imaging of the cellular dynamics of
organs and tissues in vivo [3, 4]. Therefore, we established an
advanced imaging system to visualize living bone tissues using
intravital multiphoton microscopy [5–8]. For visualization of deep
bone tissue, we selected the parietal bone of mice, which is ~80–
120 μm thick (within the range of infrared lasers), as the observa-
tion site. In this region, living bone marrow can be accessed with
minimal invasion.
To visualize mature osteoclasts (mOCs), we generated trans-
genic reporter mice expressing tdTomato, a red fluorescent pro-
tein, in the cytosol of mOCs (TRAP-tdTomato mice) [8]. To
visualize mature osteoblasts (mOBs), we also generated fluores-
cent reporter mice expressing enhanced cyan fluorescent protein
(ECFP) in the cytosol of mOBs (Col2.3-ECFP mice). To visualize
communication between mOCs and mOBs, we crossed TRAP-­
tdTomato mice with Col2.3-ECFP mice to generate TRAP-­
tdTomato/Col2.3-ECFP double fluorescently labeled mice. Using
intravital multiphoton microscopy of calvaria bone tissues of
TRAP-tdTomato/Col2.3-ECFP mice, we successfully visualized
the in vivo behavior of living mOCs and mOBs on the bone sur-
face; the imaging results suggested direct interactions between
mOCs and mOBs in vivo. In wide views of skull bones under nor-
mal conditions, mOCs and mOBs appeared to be distributed sepa-
rately, although some direct, albeit spatiotemporally limited,
mOC–mOB interactions were evident. Analysis of time-lapse
images showed that mOCs and mOBs exhibited distinct spatial
distributions. However, several mOCs in contact with mOBs dis-
played dendritic shapes and projected synapse-like structures
toward mOBs. Additionally, these interactions between mOCs and
mOBs changed dynamically according to bone homeostatic
conditions.
We recently developed pH-sensing chemical fluorescent probes
to detect localized acidification by bone-resorbing osteoclasts on
the bone surface in vivo [9, 10]. These probes are based on the
boron-dipyrromethene (BODIPY) dye and a bisphosphonate
group. BODIPY dyes have a large number of applications because
of their environmental stability, large molar absorption coefficients,
and high fluorescence quantum yields [11]. The bisphosphonate
group replaces phosphate ions in hydroxyapatite, the main compo-
nent of bone tissue, and forms a tight bond with the bone matrix.
Therefore, the bisphosphonate group facilitates delivery and fixa-
tion of the probe on bone in living animals [12]. Our recently
developed pH-probes enabled visualization of bone resorption by
osteoclasts, which led to identification of two distinct functional
states of differentiated osteoclasts: bone-resorptive [R] and non-
resorptive [N].
 Bone Imaging: Osteoclast and Osteoblast Dynamics 3

In this chapter, we describe a methodology for visualizing the

in vivo behavior of osteoclasts and osteoblasts simultaneously in
living bone tissue. In addition, we describe imaging of osteoclast
function using a pH-sensing fluorescent chemical probe.

2 Materials

2.1 Multiphoton 1. Upright multiphoton microscope (A1R-MP; Nikon) (see

Microscopy Note 1).
2.1.1 Standard Imaging 2. Water-immersion objective, 25× (APO: numerical aperture [NA],
1.1; working distance [WD], 2.0 mm; Nikon) (see Note 2).
3. Femtosecond-pulsed infrared laser (Chameleon Vision II Ti:
sapphire laser; coherent) (see Note 3).
4. External non-descanned detector (NDD) with four channels
(Nikon).
5. Dichroic and filter set: Three dichroic mirrors (458, 506, and
561 nm), and four band-pass filters (417/60 nm for the sec-
ond harmonic generation (SHG) signal, 480/40 nm for ECFP,
534/30 nm for autofluorescence, and 612/69 nm for tdTo-
mato) (Nikon) (see Note 4).
6. NIS Elements integrated software (Nikon).

2.1.2 Spectral Imaging 1. Upright multiphoton microscope (LSM 780 NLO; Carl
(See Note 5) Zeiss).
2. Water-immersion objective, 20× (W Plan-Apochromat: NA,
1.0; WD, 2.4 mm; Carl Zeiss).
3. Femtosecond-pulsed infrared laser (Chameleon Vision II Ti:
sapphire laser; coherent).
4. Internal 32-channel GaAsP spectral detectors (Carl Zeiss).
5. ZEN software (Carl Zeiss).

2.2 Mice 1. TRAP-tdTomato [8] and Col2.3-ECFP mice.

and Anesthesia 2. Isoflurane (Escain).
3. Vaporizer (inhalation device).
4. O2 bomb.
5. Anesthesia box and mask.

2.3 Intravital 1. Custom-made stereotactic stage (Fig. 1) (see Note 6).

Imaging 2. Head holder with a hexagonal window (see Note 7).
3. Shaver and hair-removal lotion.
4. Iris scissors and tweezers for mouse surgery.
5. N-Butyl cyanoacrylate glue.
4 Junichi Kikuta and Masaru Ishii

Fig. 1 Schematic illustration of calvaria bone imaging. The mouse is anesthetized

with isoflurane, the frontoparietal region of the skull bone is surgically exposed,
and its head is immobilized using the custom-made stereotactic holder. The
head holder is kept fully loaded with PBS by an infusion syringe pump

6. Ethyl-cyanoacrylate glue.
7. Infusion line.
8. Infusion syringe pump.
9. Phosphate-buffered saline (PBS) buffer, pH 7.4.
10. Electrocardiogram monitoring device.
11. Environmental dark box in which an anesthetized mouse is
warmed to 37 °C by an air heater.

2.4 Preparation 1. pH-sensing chemical fluorescent probe (Fig. 2) [10].

of the pH-Sensing 2. PBS immersion buffer, pH 7.4.
Probe
3. One 26-gauge needle.

2.5 Staining of Blood 1. Angiographic agent: Qtracker 655.

Vessels 2. One 29-gauge insulin syringes for intravenous injection.

2.6 Image 1. Image processing and analysis software: Imaris (Bitplane), NIS
Processing Elements (Nikon), and ZEN (Carl Zeiss).
and Analysis 2. After Effects software (Adobe).
 Bone Imaging: Osteoclast and Osteoblast Dynamics 5

Fig. 2 Chemical structure of the pH-sensing fluorescent chemical probe. The pH-sensing probe is based on the
boron-dipyrromethene (BODIPY) dye for visualization in low-pH environments in bone created by active osteo-
clasts; the bisphosphonate group facilitates delivery of the probe to bone tissue. Fluorescence of the probe can
be detectable under acidic condition

3 Methods

3.1 Administration 1. Dissolve 5 mg/kg of pH-sensing chemical probe (pHocas-3)

of the pH-Sensing in PBS.
Probe 2. All procedures in mice are performed under anesthesia.
3. Inject the pH-probe subcutaneously into mice daily beginning
3 days prior to imaging.
4. Perform intravital bone imaging experiments (see Subheading
3.2).

3.2 Intravital 1. Start up the multiphoton microscope and turn on the heater in
Multiphoton Imaging the environmental dark box (see Note 8).
of Calvaria Bone 2. All surgical procedures are performed under isoflurane inhala-
tion anesthesia.
3. Shave the hair and apply hair-removal lotion to the top of the
head of the mouse (see Note 9).
4. Disinfect the skin of the head using 70% ethanol.
5. Cut the skin minimally using iris scissors and then expose the
frontoparietal region of the skull.
6. Resect marginal muscles and the periosteum.
7. Apply n-butyl cyanoacrylate glue to the back of the head
holder.
8. Apply ethyl-cyanoacrylate glue to the marginal area of the
exposed bone, but not to the skull bone within the hexagonal
window.
6 Junichi Kikuta and Masaru Ishii

9. Place the head holder on the skull bone using the skull bone
suture as an anatomical landmark.
10. Wait a few minutes to allow the glue to firmly secure the skull
bone to the head holder (see Note 10).
11. Fix the head holder to the custom-made stereotactic stage
using two screws, and immobilize the mouse as tightly as pos-
sible to avoid drift secondary to respiration and pulsation
(Fig. 1) (see Note 11).
12. Set the infusion line in the groove of the head holder, and fill
the hexagonal window with PBS (see Note 12).
13. Place the mouse in the environmental dark box.
14. If necessary, intravenously inject Qtracker 655 dissolved in PBS.
15. Focus on the bone marrow cavity at an appropriate depth and
look through the ocular lenses using a mercury lamp as the
light source.
16. Change the light source from the mercury lamp to the Ti: sap-
phire laser.
17. Set the excitation wavelength, zoom ratio, z-positions, interval
time, and duration time using the microscope software (see
Note 13).
18. Observe the bone tissue by multiphoton excitation microscopy.
19. Monitor the heart rate of the mouse using an electrocardio-
gram throughout imaging (see Note 14).

3.3 Image 1. Obtain the SHG, autofluorescence, pHocas-3, ECFP, and

Processing tdTomato fluorescence spectra from the raw images using
and Analysis ZEN software by manually selecting appropriate pixels.
3.3.1 Spectral Unmixing 2. Utilize these spectral libraries for spectral unmixing
for Images Acquired algorithms.
by 32-Channel 3. Discriminate each fluorescence signal, exclude autofluores-
GaAsP Spectral Detectors cence, and create unmixed images (Fig. 3).

3.3.2 Analysis 1. Correct images for XY drift using NIS Elements or Imaris
of Intravital software.
Multiphoton Images 2. Analyze images by measuring the frequency and duration of
cell-to-cell contact using Imaris software (Fig. 4).
3. Create the movie using After Effects software.

4 Notes

1. There are two types of microscope: upright and inverted. Bone

marrow can be observed using an inverted microscope.
Multiphoton microscopes are also available from other manu-
facturers (e.g., Leica Microsystems and Olympus).
 Bone Imaging: Osteoclast and Osteoblast Dynamics 7

Fig. 3 Visualization of the bone-resorptive function of mature osteoclasts using a

pH-sensing fluorescent probe. Representative images of the calvaria of TRAP-­
tdTomato mice treated with a pH-probe, showing sites of local bone resorption.
Red, mature osteoclasts expressing TRAP-tdTomato; green, fluorescence signals
from areas of high H+ concentration; blue, bone tissue. Arrowheads and asterisks
represent bone-resorptive and non-resorptive osteoclasts, respectively. Scale
bar, 40 μm

Fig. 4 Visualization of living mature osteoclasts and osteoblasts on bone surface

by intravital multiphoton microscopy. Representative image of the calvaria of
TRAP-tdTomato/Col2.3-enhanced cyan fluorescent protein (ECFP) mice under
control conditions. Red, mature osteoclasts (mOCs) expressing TRAP-tdTomato;
cyan, mature osteoblasts (mOBs) expressing Col2.3-ECFP. Arrowheads represent
mOB–mOC interactions. Scale bar, 40 μm
8 Junichi Kikuta and Masaru Ishii

2. Objective lenses with a higher NA and longer WD are

desirable.
3. A femtosecond-pulsed infrared laser is also available from
Spectra-Physics (MaiTai).
4. The dichroic and filter set required depends on the fluorescent
proteins and dyes used.
5. We utilize a Carl Zeiss upright multiphoton microscope with
internal 32-channel GaAsP spectral detectors when using
simultaneous multiple fluorescent labels. This enables acquisi-
tion of the entire spectrum of each label in one scan, which is
then used to create unmixed images.
6. The custom-made stereotactic stage is composed of a 10-mm-
thick metal plate, on which two cylinders with screw holes are
placed. The head holder can be fixed to this stage with two
stainless screws.
7. The head holder has one recess, the curvature radius of which
is 28 mm. The center of the recess has a hexagonal window.
The head holder is 3 mm in thickness and 15 g in weight.
8. Some time is required for the laser and temperature to
stabilize.
9. Remove as much hair as possible to prevent hair (which is
autofluorescent) entering the visual field.
10. Prevent glue from contaminating the visual field because some
glues are autofluorescent.
11. Do not fasten too tightly to avoid injuring the mouse.
12. The head holder is kept fully loaded with PBS by an infusion
syringe pump.
13. The excitation wavelength of 940 nm is used to simultaneously
excite ECFP, tdTomato, and pHocas-3. For an example of
intravital time-lapse bone imaging, image stacks were collected
at 3 μm vertical steps at a depth of 50–150 μm below the skull
bone surface with 2.0× zoom, 512 × 512 X–Y resolution, and
a time resolution of 5 min.
14. The heart rate is used as a guide to adjust the anesthetic gas
concentration.

References

1. Hattner R, Epker BN, Frost HM (1965) multitude of signals within the basic multicel-
Suggested sequential mode of control of lular unit. Bonekey Rep 3:481
changes in cell behavior in adult bone remodel- 3. Cahalan MD, Parker I, Wei SH, Miller MJ
ing. Nature 206:489–490 (2002) Two-photon tissue imaging: seeing the
2. Sims NA, Martin TJ (2014) Coupling the immune system in a fresh light. Nat Rev
activities of bone formation and resorption: a Immunol 2:872–880
 Bone Imaging: Osteoclast and Osteoblast Dynamics 9

4. Germain RN, Miller MJ, Dustin ML, 8. Kikuta J et al (2013) Dynamic visualization of
Nussenzweig MC (2006) Dynamic imaging of RANKL- and Th17-mediated osteoclast func-
the immune system: progress, pitfalls and tion. J Clin Invest 123:866–873
promise. Nat Rev Immunol 6:497–507 9. Kowada T et al (2011) In vivo fluorescence
5. Ishii M et al (2009) Sphingosine-1-phosphate imaging of bone-resorbing osteoclasts. J Am
mobilizes osteoclast precursors and regulates Chem Soc 33:17772–17776
bone homeostasis. Nature 458:524–528 10. Maeda H et al (2016) Real-time intravital
6. Ishii M, Kikuta J, Shimazu Y, Meier-­ imaging of pH variation associated with osteo-
Schellersheim M, Germain RN (2010) clast activity. Nat Chem Biol 12:579–585
Chemorepulsion by blood S1P regulates osteo- 11. Loudet A, Burgess K (2007) BODIPY dyes
clast precursor mobilization and bone remodel- and their derivatives: syntheses and spectro-
ing in vivo. J Exp Med 207:2793–2798 scopic properties. Chem Rev 107:4891–4932
7. Kikuta J et al (2013) Sphingosine-1-phosphate-­ 12. Kozloff KM, Volakis LI, Marini JC, Caird MS
mediated osteoclast precursor monocyte migra- (2010) Near-infrared fluorescent probe traces
tion is a critical point of control in the bisphosphonate delivery and retention in vivo.
anti-bone-resorptive action of active vitamin J Bone Miner Res 25:1748–1758
D. Proc Natl Acad Sci U S A 110:7009–7013
 Chapter 2

Intravital Imaging of Mouse Bone Marrow: Hemodynamics

and Vascular Permeability
Yookyung Jung, Joel A. Spencer, Anthony P. Raphael, Juwell W. Wu,
Clemens Alt, Judith R. Runnels, Briaira Geiger, and Charles P. Lin

Abstract
The bone marrow is a unique microenvironment where blood cells are produced and released into the
circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought
to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden,
Nature 505:327–334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make
close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originat-
ing from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit differ-
ent vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al.,
Nature 532:323–328, 2016; Spencer et al., Nature 508:269–273, 2014). Here, we describe a method to
quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial
bone marrow using intravital microscopy.

Key words Bone marrow blood vessel, Arterioles, Sinusoids, Permeability, Hemodynamics, Flow
speed, Blood vessel diameter, Mouse restraint, Intravital imaging

1 Introduction

The microanatomic landscape of the bone marrow is dominated by

the presence of a dense vascular network. The vasculature makes
up approximately 25–30% of the marrow space by volume [1, 2],
with sinusoidal blood vessels being the most prominent vessel type.
However, the bone marrow vasculature is heterogeneous [3, 4],
and the heterogeneity is particularly notable when imaging the
local hemodynamics in real time [1, 5]. Compared to arteriolar
vessels, the flow speed and sheer rate in sinusoidal vessels are much
lower [1, 5], and vascular permeability is significantly higher [5].
Increased vascular permeability has been shown to increase the
level of reactive oxygen species (ROS) in the surrounding cells and

Yookyung Jung and Joel A. Spencer contributed equally to this work.

Masaru Ishii (ed.), Intravital Imaging of Dynamic Bone and Immune Systems: Methods and Protocols, Methods in Molecular Biology,
vol. 1763, https://doi.org/10.1007/978-1-4939-7762-8_2, © Springer Science+Business Media, LLC 2018

11
12 Yookyung Jung et al.

to impact the migration and differentiation of hematopoietic stem

and progenitor cells (HSPCs) [5]. Here, we present a protocol
for measuring hemodynamics and permeability of arterioles and
sinusoids in mouse bone marrow using video rate laser scanning
confocal/two-photon microscopy [6–8]. Video rate laser scanning
is achieved by a polygon-based fast scanning mechanism that allows
full-frame (500 × 500 pixels) image acquisition at 30 frames per
second. Higher frame rates can be acquired at a reduced frame size
(120 frames per second at 500 × 125 pixels). The rapid scanning
enables individual hemodynamic and vessel permeability measure-
ments based on fluorescently labeled circulating blood cells and
vascular dye leakage, respectively. Using this protocol, we could
measure flow speeds up to 10 mm/s in individual blood vessels
and simultaneously record vascular permeability in the same vessels
within the first minute after dye injection. We also present concur-
rent methods for stabilizing the mouse for imaging and injection
of cells and dyes on stage using a custom-built catheter.

2 Materials

2.1 Mouse Any strain of mice can be used. Care should be taken to minimize
signal overlap with the vascular dyes for permeability studies if co-­
injecting fluorescently labeled cells or using transgenic fluorescent
mouse models. We typically use C57BL/6, which is the most com-
mon background strain for our studies. Animals were maintained
in the animal facilities of Massachusetts General Hospital in com-
pliance with institutional guidelines, and all animal studies were
approved by the Subcommittee on Research Animal Care of the
institution.
The high resolution imaging demands minimal mouse move-
ment during imaging caused by breathing and other movement
artifacts (see Note 1). Adequate mouse restraint can be achieved by
modifying a 50 mL conical tube as described in [4]. However, with
improved access to rapid-prototyping and related CAD software,
more precise restraints can be fabricated. Our custom-built, 3D
printed mouse restraint was designed using Autodesk® Inventor
Professional 3D CAD software (Autodesk®, USA) and printed on a
Fortus 380mc (Stratasys, USA) using ASA filament (see Note 2).

2.2 Fluorescence Fluorescently labeled RBCs are used for flow speed measurement.
Labeling of Red Blood Here we assumed that the local blood flow speed is equal to the
Cells (RBCs) average speed of RBCs in the vasculature.
Solutions and heparin-coated microcentrifuge tubes should be
prepared immediately before RBC labeling.
1. Fluorescent cell label CFDA-SE: thaw one vial of 500 μg
CFDA-SE (V12883, Invitrogen) and solubilize in 90 μL
DMSO.
 Hemodynamics and Permeability of Bone Marrow Blood Vessels 13

2. Labeling solution A (“Soln A”): 90 mL of PBS (without

calcium and magnesium) supplemented with 1 g/L glucose
and 0.1% bovine serum albumin (BSA). Keep 60 mL at room
temperature, warm 30 mL to 37 °C.
3. Labeling solution B (“Soln B”): 48 mL PBS (without calcium
and magnesium), warm to 37 °C.
4. Wash solution (“Soln W”): 30 mL PBS (without calcium and
magnesium) supplemented with 1 g/L glucose. Keep at room
temperature.
5. Donor mouse: we label 1.2 × 109 RBCs for a final yield of
>0.75 × 109 cells. In our experience, a single facebleed from an
adult unanesthetized C57BL/6 mouse provides sufficient
RBCs.
6. Microcentrifuge tube for blood collection: add 200 μL
100 unit/mL heparin into tube and vortex briefly to coat.
7. Any BSA stock solution (concentration varies by
manufacturer).

2.3 Fluorescence Vascular dyes are used for flow speed measurement, vessel diameter
Labeling of the measurement, and vascular permeability measurement.
Vasculature
1. Large molecular weight fluorescent dye-dextran conjugates:
10 mg/mL 70 kDa Rhodamine B-dextran, 10 mg/mL
70 kDa Texas red-dextran, 10 mg/mL 70 kDa
FITC-dextran.

2.4 Custom-Built 1. 30½ gauge needle.

Catheter
2. Tygon microbore tubing, 0.010 (ID) × 0.030 (OD) in.
3. ½ cc insulin syringe with 29 G × ½ in. needle.

3 Methods

3.1 Preparing RBCs are fluorescently labeled ex vivo and injected into recipient
Fluorescently Labeled mouse 2–3 days before imaging to allow time for the bone marrow
RBCs to re-establish its equilibrium hemodynamic state.
1. Perform facial vein blood collection on donor mouse and col-
lect the blood in heparin-coated microcentrifuge tube. Animal
anesthesia (if used) should be non-intravenous, such as isoflu-
rane. Avoid ketamine and xylazine.
2. Transfer the heparinized blood suspension into 12 mL of Soln
A at room temperature. Centrifuge at 500 × g for 6 min.
Discard the suspension, which should be opaque with a faint
red shade. The cell pellet should be bright red.
14 Yookyung Jung et al.

3. Wash the RBCs two more times with 1.5 mL Soln A at room
temperature.
4. Perform cell count. Resuspend 1.2 × 109 RBCs in 12 mL Soln
A (37 °C) at 1 × 108 cells/mL. Keep the cells at 37 °C.
5. Add the 90 μL CFDA-SE DMSO stock into Soln B. Vortex to
mix and split the solution evenly into two 50 mL centrifuge
tubes (24 mL each).
6. Mix 6 mL of the RBC suspension from step 4 into each of the
two 50 mL centrifuge tubes prepared in step 5. Label at 37 °C
for 12 min.
7. When labeling is complete, introduce bovine serum albumin
stock solution such that BSA concentration is 0.1% in each of
the two 50 mL centrifuge tubes. Centrifuge.
8. Wash each cell pellet in 30 mL Soln A at room temperature.
Centrifuge.
9. Resuspend each cell pellet in 15 mL Soln W at room tempera-
ture and combine the two suspensions into one centrifuge
tube. Perform cell count and centrifuge.
10. Resuspend the labeled RBCs in 180 μL saline for retro-orbital
or tail vein injection. The RBC suspension should retain the
same bright red hue as in blood; do not inject cells that look
“rusty” as they will be cleared by circulation.

3.2 Mouse Restraint 1. Anesthesia is induced using 4% isoflurane in oxygen with a

and Preparation maintenance rate of 1–3%. Induction is achieved in a separate
chamber prior to mounting the mouse in the restraint.
2. For minimal motion, the mouse is first restrained by the bite-­
bar, followed by the nose cone and lastly the side skull
restraints. The contact points on the side of the skull should be
between the eye socket and ears, typically in the region of the
squamosal bones (see Fig. 1).
3. Following appropriate restraint, the skull is prepared after
scalp incision [7].

3.3 Intravital The protocol for performing intravital optical imaging of the bone
Imaging of Calvarial marrow has been well described [6–8]. It is summarized as follows.
Bone Marrow Figure 2a shows the cross-sectional view of the calvarial bone of a
mouse. As the bone overlying the marrow cavity is typically
50–70 μm thick, the bone marrow can be accessed optically after a
simple skin flap surgery to expose the underlying bone surface.
The thickness of the marrow cavity can vary from tens of microm-
eters to a couple of hundred micrometers (Fig. 2a and b), where
the low intensity region in between the bone layers is the bone
marrow. Using the intrinsic second harmonic signal to visualize
 Hemodynamics and Permeability of Bone Marrow Blood Vessels 15

Fig. 1 3D printed mouse holder for intravital optical imaging. (a) Mouse holder.
Red arrows indicate the nose cone and bite-bar which are anchor points for
securing the mouse head. Red dots are prongs that fix squamosal bones of the
mouse. (b) Side view of mouse skull. Red arrows and dots of (a) are shown at the
corresponding sites [6]

the bone, we image the bone marrow cavities located in the central
region of the skull around the sagittal and coronal sutures, which
encompass an area approximately 3–6 by 6–8 mm (see Fig. 2c and
Note 3). An example image of a bone marrow cavity of a nestin-­
GFP mouse is shown in Fig. 2d, with the bone signal in blue, GFP
in green, hematopoietic stem and progenitor cell in red, and blood
vessels in grey. The cell is overlaid on the pre-acquired nestin-GFP,
blood vessels, and bone images.

3.4 Imaging Labeled The blood flow speed is measured by tracking the frame-to-frame
RBC and Calculating displacement of individual RBCs using high frame rate imaging.
Flow Speed 750–800 million fluorescently labeled RBCs are injected into a
recipient mouse 2–3 days before imaging. Immediately prior to
imaging, a fluorescent vascular dye (e.g., Rhodamine-B-dextran) is
injected allowing the visualization of the blood vessels that map
the “paths” of RBC displacement. For accurate flow speed mea-
surement, the average velocity of a minimum of five RBCs is used.
The detailed protocol for determining RBC and blood flow
speed in vivo is as follows:
16 Yookyung Jung et al.

Fig. 2 Intravital optical imaging of the bone marrow through the calvarial bone. (a) Cross-sectional view of a
mouse skull (from Henkelman M, Micro-CT images of mouse skull. http://www.stlfinder.com/model/mouse-
skull-(from-micro-ct)) and optical access into the bone marrow between calvarial bone layers. (b) Intensity
profile of the dotted yellow line in (a) shows two layers of calvarial bone and bone marrow between them. (c)
Top view of a mouse skull. Optical imaging area is shown as a dotted red rectangle that contains coronal and
sagittal sutures [6]. (d) Example of in vivo optical imaging of the bone marrow [9]. Nestin-GFP (green), hema-
topoietic stem and progenitor cell (red), blood vessels (grey), and bone (blue) are displayed. The cell is overlaid
on the pre-acquired nestin-GFP, blood vessels, and bone images. Scale bar is 50 μm

1. Deliver 40 μL of 10 mg/mL 70 kDa Rhodamine B-dextran

(ThermoFisher Scientific) to a labeled RBC recipient mouse
by retro-orbital or tail vein intravenous (i.v.) injection for
vascular labeling.
2. Perform simultaneous in vivo imaging of bone, blood vessels,
and labeled RBC. Imaging conditions are as follows:
●● Image size: 500 × 125 pixels.
●● Imaging speed: 120 frames/s.
●● Bone: 840 nm femtosecond laser excitation, ~40 mW of
power at the sample, 80 MHz repetition rate, MaiTai,
 Hemodynamics and Permeability of Bone Marrow Blood Vessels 17

Fig. 3 Flow of labeled RBCs in the bone marrow blood vessels. Blue: second harmonic generation signal from
bone collagen. Red: blood vessels. Green: labeled RBCs. Scale bars are 50 μm. (a) Example image showing
overlay of labeled RBCs at several consecutive frames. (b) Flow of a single RBC in an arteriole. (c) Flow of a
single RBC in a sinusoid

Spectra Physics, Collection of the second harmonic genera-

tion from collagen in bone.
●● CFSE-labeled RBC: 491 nm continuous wave (CW) laser
excitation, ~1 mW of power at the sample, Dual Calypso,
Cobolt, Collection of confocal fluorescence at the spectral
range of 509–547 nm.
●● Blood vessel: 561 nm CW laser excitation, Jive, Cobolt,
~1 mW of power at the sample, Collection of confocal
fluorescence at the spectral range of 573–613 nm.
3. Calculate the speed of the blood flow using the following
equation (see Note 4):
Total distance traveled by RBC/Time (= number of
frames × 1/120 s).
The flow of labeled RBCs in the bone marrow blood vessels is
shown (Fig. 3). The labeled RBCs at several consecutive frames are
overlaid on the vascular image. Dotted and solid arrows show the
direction of flow and the positions of RBCs at each frame,
respectively.
Arterioles with small diameters (5–10 μm) show fast flow
(>1.5 mm/s) (Fig. 3b), and sinusoids with large diameter (>20 μm)
show slow flow (<1 mm/s) (Fig. 3c).

3.5 Blood Vessel Based on the flow speed and vessel diameter (Fig. 4), the majority
Diameter of bone marrow blood vessels can be categorized into two distinct
Measurement groups (Fig. 4f), those with high flow speed and small diameter
18 Yookyung Jung et al.

Fig. 4 Flow speed and diameter of arterioles and sinusoids. (a) Two-photon image of the blood vessels in the
calvarial bone marrow. Numbers in red and blue indicate the blood vessel identification (ID). Scale bar is
50 μm. (b, c) Individual blood vessel flow speeds from arterioles and sinusoids, respectively. (d, e) Individual
blood vessel diameters from arterioles and sinusoids, respectively. (f) Scatterplot of flow speed as a function
of diameter. Two distinct groups of “high flow speed and small diameter” vs “low flow speed and large diam-
eter” can be found (data from 70 blood vessels, n = 6 mice). Specific site of the individual blood vessels with
ID numbers in (b–e) can be found in (a)

(arterioles) vs those with low flow speed and large diameter

(sinusoids). These results are consistent with published values [1].
The protocol for blood vessel diameter measurement is as
follows:
1. Deliver 40 μL of 10 mg/mL 70 kDa Rhodamine B-dextran
(ThermoFisher Scientific) by retro-orbital or tail vein intrave-
nous (i.v.) injection for vascular labeling.
2. Acquire two-photon excitation fluorescence (at the spectral
range of 550–680 nm) image of the vessels in the calvarial bone
marrow immediately after injecting the vascular dye with femto-
 Hemodynamics and Permeability of Bone Marrow Blood Vessels 19

second laser source (~40 mW of power at the sample, 80 MHz

repetition rate, 840 nm, MaiTai, Spectra Physics). Example
image shown in Fig. 4a.
3. Measure the diameter of the blood vessels using image analysis
software, for example ImageJ or Fiji software [10]. Repeated
measurements are taken and the averaged value of the measured
diameters is acquired.

3.6 Preparation Because of the high permeability of the bone marrow vasculature,
of Custom-Built it is necessary to perform dye injection while the mouse is on stage,
Catheter for Vascular so that the early dynamics of dye leakage immediately after injection
Imaging can be recorded and analyzed.
and Permeability
Measurement

3.6.1 Catheter 1. Cut 30½ gauge needle by using forceps (Fig. 5(1)).
Preparation 2. Insert the cut needle into 0.030 in. flexible plastic tube
(Fig. 5(2)).
3. Insert a needle of an insulin syringe into the other end of the
plastic tube (Fig. 5(3)).

Fig. 5 Preparation of home-built catheter for repeated vascular imaging and per-
meability measurement
20 Yookyung Jung et al.

4. Repeated bone marrow vascular imaging and permeability

measurements can be performed by using the home-built
catheter.
5. In case multiple measurements of permeability are necessary,
vasculature dyes with distinct two-photon emission spectrum
should be used to prevent the signal leaking between the
detection channels (see Note 5).

3.6.2 Quantitative 1. Perform video rate two-photon imaging of bone marrow vas-
Measurement culature immediately after injection of vascular dye.
of Permeability 2. Frame-to-frame misalignment of the images can exist because
of residual movement artifacts from breathing (Fig. 6a).
3. Series of raw images in Fig. 6a are registered and aligned by
either using Template Matching plugin of ImageJ or Matlab
code utilizing the command, normxcorr2, which performs
normalized two-dimensional cross-correlation [11, 12]. Ten

a b

c d 1 (Sinusoid)
40
2 (Arteriole)

30
Intensity (a.u.)

20

10

0
0 5 10 15 20 25 30 35
Time (second)

Fig. 6 Process to measure the rate of vascular dye leakage. (a) Series of two-photon raw vascular images with
1/30 s of time interval. Misalignment of the images exists because of residual movement artifacts. (b) Raw
images are registered and aligned. Ten consecutive frames are frame-averaged to reduce the background noise
signal. (c) Regions to measure the dye leakage are displayed as yellow rectangles labeled with 1 (next to sinu-
soid) and 2 (next to arteriole). (d) Average intensity in the region 1 and 2 of (c) is plotted as a function of time.
The linear fits are shown as red lines. The slope of this linear fit is the rate of vascular dye leakage, dI/dt
Exploring the Variety of Random
Documents with Different Content
commensurate reward (not wholly undeserved) for the mental labor
involved in its successful evolution.
Its simplicity is such that it can be manipulated by any intelligent child,
and its price, by comparison with its remedial virtues, is insignificant. With
this perfected apparatus, and the J. B. L. antiseptic tonic, any parent can
constitute himself the physician of his family, and by following the
directions for the treatment of the various diseases described in this work,
can successfully combat them—and all at a trifling cost. But more than that,
he can, by periodical use of it, so improve the physical condition of himself
and family, that they will forget what sickness is, and rejoice in that
exhilaration of spirit that only comes with perfect health.
My system of treatment is true in philosophy, in harmony with Nature,
and thoroughly rational in practice.
INDEX TO TREATMENT OF DISEASE.
A, B, C, D, E, F, G, H, I, L, M, N, O, P, R, S, T, U, W

PAGE
Anæmia, 159
Anteversion, 186
Appendicitis, 176
Asiatic Cholera, 181
Asthma, 184
Bilious Fever, 170
Blood Poisoning, 161
Bronchitis, 184
Catarrh, 164
Cholera Infantum, 204
Cholera Morbus, 182
Common Colds, 187
Constipation, 188
Consumption, 102
Croup, 204
Diabetes, or Diabetes Mellitus, 199
Diarrhœa, 173
Diphtheria, 205
Diseases of the Kidneys, 180
Diseases of the Liver, 178
Diseases of the Nerves, 173
Diseases of the Skin, 179
Dropsy, 176
Dysentery, 172
Dyspepsia, 100
Epilepsy, or Falling Sickness, 192
Erysipelas, 165
Fistula, 203
 Gonorrhea, 193
Gall Stones, 208
Headache, 175
Heart Disease, 158
Hernia, or Rupture, 194
Inebriety, 194
Infantile, Convulsions, or Fits, 207
Inflammation of the Breast, 201
La Grippe, 171
Locomotor Ataxia, 200
Lost Manhood, 197
Measles, 207
Milk Fever, 202
Nursing Mothers, 200
Obesity, 196
Paralysis, or Palsy, 190
Peritonitis, 182
Piles, or Hemorrhoids, 189
Pneumonia, 183
Puerperal Swelled Leg, 202
Retroversion, 186
Rheumatism, 167
Scarlet Fever, 204
Small-pox, 206
Sore Nipples, 202
Typhoid Fever, 168
Uterine Displacement, 185
Worms in the Intestines, 207

IF YOU SUFFER
FROM ROUGH, SCALY OR CRACKED SKIN
If You Value a Good Complexion
 Dr. Tyrrell’s Health Soap
Effectually Disposes of Troubles
IT IS REFRESHING, PURIFYING, INVIGORATING
Among the necessities of life there is one to which few people pay the
attention they ought, and that is Soap. Yet it is undoubtedly a most
important matter, for the skin is a very delicate and sensitive organ, and the
constant application of impure or inferior Soaps injures its texture, and
gives rise to numerous cutaneous troubles. Most people are content, so long
as it appeals to the eye and the sense of smell, without stopping to consider
that perfumes may be employed to hide defects.
Dr. Tyrrell has given this matter long and profound consideration and
now offers to the public a SOAP that leaves nothing to be desired. It is not
only absolutely free from any deleterious substance, but is a perfect
antiseptic and medicine soap. Its use thoroughly cleanses and invigorates
the skin, keeps it soft, flexible and healthy, and effectually prevents rough,
cracked and scaly conditions. It is invaluable for TAN, FRECKLES,
SUNBURN, Etc., and is a perfect hygienic safeguard against cutaneous
disorders. It is a positive pleasure to use it for the toilet or bath, as it leaves
such a grateful, refreshing after-effect.
As a SHAVING SOAP it is unequalled, absolutely preventing those
disagreeable results that frequently follow the use of impure soap.
25 Cents Per Cake
MANUFACTURED SOLELY BY
CHARLES A. TYRRELL, M. D.
Proprietor of
TYRRELL’S HYGIENIC INSTITUTE
134 West 65th Street, New York City

The J. B. L. Antiseptic Tonic

should always be used when introducing water into the intestines. The use
of this preparation renders the water completely sterile, unless it be
notoriously impure. Such water should never be used. But the Antiseptic
Tonic possesses another important property which is most valuable in cases
of Constipation. For it acts as an admirable tonic on the muscular coat of
the colon, strengthening it and restoring it to normal. For these reasons it is
invaluable. Owing to the importance of using the tonic I have arranged to
make it as inexpensive as possible, and am prepared to furnish it (to users of
the Cascade only) in one pound air-proof cans at the price of $1.00, by mail
twenty cents extra. You can buy it at your druggist and save mail charges.
Charles A. Tyrrell, M. D.,
134 West 65th Street,
New York City.

You’re Not Healthy Unless You’re Clean

Inside
And the one way to real internal cleanliness—by which you are
protected against ninety per cent, of all human ailments—is through proper
internal bathing, with plain warm water.
There is nothing unusual about this treatment—no drugs, no dieting—
nothing but the correct application of Nature’s own cleanser. But only since
the invention of the “J. B. L. Cascade” has a means for proper internal
bathing existed.
Only one treatment is known for actually cleansing the colon without
the aid of elaborate surgical apparatus. This is The Internal Bath by means
of the “J. B. L. Cascade.”
Prof. Metchnikoff, Europe’s leading authority on intestinal conditions, is
quoted as saying that, if the colon and its poisonous contents were removal,
people would live in good health to twice the present average of human life.
Dr. A. Wilford Hall, Ph.D., LL.D., and W. E. Forest, B.D., M.D., two
world-famous authorities on internal bathing, are among the thousands of
physicians who have given their hearty and active endorsement and support
to the “J. B. L. Cascade” treatment.
Fully half a million men and women and children now use this real boon
to humanity—most of them in accordance with their doctor’s orders.
LET DR. TYRRELL ADVISE YOU
Dr. Tyrrell is always very glad of an opportunity to consult freely with any one who
writes him-and at no expense or obligation whatever. Describe your case to him and he
gives you his promise that you will learn facts about yourself which you will realize are of
vital importance. You will also receive his book, “The What, The Why, The Way,” which
is a most interesting treatise on internal bathing. Consultation with Dr. Tyrrell involves
no obligation.

CHARLES A. TYRRELL, M. D.,

134 West 65th Street, New York.

Sufferers From Catarrh There Is Glorious News For

You
No matter how much you may suffer from that most distressing and
inconvenient complaint, a speedy and effective release from your sufferings
is now offered to you

The J. B. L. Catarrh Balsam

Is one of those sterling preparations whose healing effects are quickly
realized on the first trial. It is intended to be used in connection with the
flushing treatment, and the two used in conjunction.
SELDOM FAILS TO EFFECT RELIEF
Catarrh is first caused by inflammation of the membrane of the nasal
cavities and air passages, which is followed by ulceration, when nature, in
order to shelter this delicate tissue, and protect the olfactory nerves, throws
a tough membrane over the ulcerated condition.
Flushing the Colon lays the foundation for recovery, but the membrane
must be removed, and for that purpose the J. B. L. Catarrh Remedy is
without an equal.
It is composed of several kinds of oils, and gently, but effectually
removes the membrane that nature has built over the inflamed parts, while
its emollient character soothes and allays the inflammation. These drugs are
not absorbed into the system, but act only locally.
THE MOST OBSTINATE CASE WILL READILY YIELD
TO THIS TREATMENT
The price is One Dollar per bottle, which, in view of its marvellous
efficiency, is a veritable gift, and with each bottle we furnish an inhaler
specially manufactured for the purpose. Two bottles will usually effect
relief—though one has been frequently known to do so in mild cases—but
in the event of any one taking six bottles without being benefited we will
forfeit
ONE HUNDRED DOLLARS,
now deposited in the Lincoln Trust Co. of New York, if they can honestly
make oath that they have faithfully used the remedy according to the
directions and have received no benefit from it.
You cannot afford to neglect this opportunity of ridding yourself of this
most distressing complaint, which, if neglected, too often leads to
Consumption.
DELAYS ARE DANGEROUS
————
CHARLES A. TYRRELL, M. D., PROPRIETOR OF
TYRRELL’S HYGIENIC INSTITUTE
134 WEST 65TH STREET, NEW YORK

Typographical errors corrected by the etext transcriber:

their disincilination to adopt=> their disinclination to adopt {pg 29}
Prof. I. I. Metchinkoff=> Prof. I. I. Metchnikoff {pg 43}
to what school be belongs=> to what school he belongs {pg 51}
an unmistakaby valuable=> an unmistakably valuable {pg 68}
are frequntly guilty=> are frequently guilty {pg 92}
to his pirmitive condition=> to his primitive condition {pg 104}
populated with phagocyctes (white blood corpuscles)=> populated with
phagocytes (white blood corpuscles) {pg 108}
an expert dietetician=> an expert dietician {pg 128}
We habituate ouselves=> We habituate ourselves {pg 151}
Too little attenion=> Too little attention {pg 156}
Griping pains=> Gripping pains {pg 181}
the bony constitutents=> the bony constituents {pg 201}
most effiacious in stimulating=> most efficacious in stimulating {pg
210}
 *** END OF THE PROJECT GUTENBERG EBOOK THE ROYAL ROAD
TO HEALTH; OR, THE SECRET OF HEALTH WITHOUT DRUGS ***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying
copyright royalties. Special rules, set forth in the General Terms of
Use part of this license, apply to copying and distributing Project
Gutenberg™ electronic works to protect the PROJECT GUTENBERG™
concept and trademark. Project Gutenberg is a registered trademark,
and may not be used if you charge for an eBook, except by following
the terms of the trademark license, including paying royalties for use
of the Project Gutenberg trademark. If you do not charge anything
for copies of this eBook, complying with the trademark license is
very easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the free

distribution of electronic works, by using or distributing this work (or
any other work associated in any way with the phrase “Project
Gutenberg”), you agree to comply with all the terms of the Full
Project Gutenberg™ License available with this file or online at
www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand, agree
to and accept all the terms of this license and intellectual property
(trademark/copyright) agreement. If you do not agree to abide by all
the terms of this agreement, you must cease using and return or
destroy all copies of Project Gutenberg™ electronic works in your
possession. If you paid a fee for obtaining a copy of or access to a
Project Gutenberg™ electronic work and you do not agree to be
bound by the terms of this agreement, you may obtain a refund
from the person or entity to whom you paid the fee as set forth in
paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only be

used on or associated in any way with an electronic work by people
who agree to be bound by the terms of this agreement. There are a
few things that you can do with most Project Gutenberg™ electronic
works even without complying with the full terms of this agreement.
See paragraph 1.C below. There are a lot of things you can do with
Project Gutenberg™ electronic works if you follow the terms of this
agreement and help preserve free future access to Project
Gutenberg™ electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright law
in the United States and you are located in the United States, we do
not claim a right to prevent you from copying, distributing,
performing, displaying or creating derivative works based on the
work as long as all references to Project Gutenberg are removed. Of
course, we hope that you will support the Project Gutenberg™
mission of promoting free access to electronic works by freely
sharing Project Gutenberg™ works in compliance with the terms of
this agreement for keeping the Project Gutenberg™ name associated
with the work. You can easily comply with the terms of this
agreement by keeping this work in the same format with its attached
full Project Gutenberg™ License when you share it without charge
with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside the
United States, check the laws of your country in addition to the
terms of this agreement before downloading, copying, displaying,
performing, distributing or creating derivative works based on this
work or any other Project Gutenberg™ work. The Foundation makes
no representations concerning the copyright status of any work in
any country other than the United States.

1.E. Unless you have removed all references to Project Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project Gutenberg™
work (any work on which the phrase “Project Gutenberg” appears,
or with which the phrase “Project Gutenberg” is associated) is
accessed, displayed, performed, viewed, copied or distributed:
 This eBook is for the use of anyone anywhere in the United
States and most other parts of the world at no cost and with
almost no restrictions whatsoever. You may copy it, give it away
or re-use it under the terms of the Project Gutenberg License
included with this eBook or online at www.gutenberg.org. If you
are not located in the United States, you will have to check the
laws of the country where you are located before using this
eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is derived

from texts not protected by U.S. copyright law (does not contain a
notice indicating that it is posted with permission of the copyright
holder), the work can be copied and distributed to anyone in the
United States without paying any fees or charges. If you are
redistributing or providing access to a work with the phrase “Project
Gutenberg” associated with or appearing on the work, you must
comply either with the requirements of paragraphs 1.E.1 through
1.E.7 or obtain permission for the use of the work and the Project
Gutenberg™ trademark as set forth in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is posted

with the permission of the copyright holder, your use and distribution
must comply with both paragraphs 1.E.1 through 1.E.7 and any
additional terms imposed by the copyright holder. Additional terms
will be linked to the Project Gutenberg™ License for all works posted
with the permission of the copyright holder found at the beginning
of this work.

1.E.4. Do not unlink or detach or remove the full Project

Gutenberg™ License terms from this work, or any files containing a
part of this work or any other work associated with Project
Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute this

electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1
with active links or immediate access to the full terms of the Project
Gutenberg™ License.

1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if you
provide access to or distribute copies of a Project Gutenberg™ work
in a format other than “Plain Vanilla ASCII” or other format used in
the official version posted on the official Project Gutenberg™ website
(www.gutenberg.org), you must, at no additional cost, fee or
expense to the user, provide a copy, a means of exporting a copy, or
a means of obtaining a copy upon request, of the work in its original
“Plain Vanilla ASCII” or other form. Any alternate format must
include the full Project Gutenberg™ License as specified in
paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™ works
unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or providing

access to or distributing Project Gutenberg™ electronic works
provided that:

• You pay a royalty fee of 20% of the gross profits you derive
from the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
 about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt
that s/he does not agree to the terms of the full Project
Gutenberg™ License. You must require such a user to return or
destroy all copies of the works possessed in a physical medium
and discontinue all use of and all access to other copies of
Project Gutenberg™ works.

• You provide, in accordance with paragraph 1.F.3, a full refund of

any money paid for a work or a replacement copy, if a defect in
the electronic work is discovered and reported to you within 90
days of receipt of the work.

• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.

1.E.9. If you wish to charge a fee or distribute a Project Gutenberg™

electronic work or group of works on different terms than are set
forth in this agreement, you must obtain permission in writing from
the Project Gutenberg Literary Archive Foundation, the manager of
the Project Gutenberg™ trademark. Contact the Foundation as set
forth in Section 3 below.

1.F.

1.F.1. Project Gutenberg volunteers and employees expend

considerable effort to identify, do copyright research on, transcribe
and proofread works not protected by U.S. copyright law in creating
the Project Gutenberg™ collection. Despite these efforts, Project
Gutenberg™ electronic works, and the medium on which they may
be stored, may contain “Defects,” such as, but not limited to,
incomplete, inaccurate or corrupt data, transcription errors, a
copyright or other intellectual property infringement, a defective or
damaged disk or other medium, a computer virus, or computer
codes that damage or cannot be read by your equipment.

1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except for

the “Right of Replacement or Refund” described in paragraph 1.F.3,
the Project Gutenberg Literary Archive Foundation, the owner of the
Project Gutenberg™ trademark, and any other party distributing a
Project Gutenberg™ electronic work under this agreement, disclaim
all liability to you for damages, costs and expenses, including legal
fees. YOU AGREE THAT YOU HAVE NO REMEDIES FOR
NEGLIGENCE, STRICT LIABILITY, BREACH OF WARRANTY OR
BREACH OF CONTRACT EXCEPT THOSE PROVIDED IN PARAGRAPH
1.F.3. YOU AGREE THAT THE FOUNDATION, THE TRADEMARK
OWNER, AND ANY DISTRIBUTOR UNDER THIS AGREEMENT WILL
NOT BE LIABLE TO YOU FOR ACTUAL, DIRECT, INDIRECT,
CONSEQUENTIAL, PUNITIVE OR INCIDENTAL DAMAGES EVEN IF
YOU GIVE NOTICE OF THE POSSIBILITY OF SUCH DAMAGE.

1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you

discover a defect in this electronic work within 90 days of receiving
it, you can receive a refund of the money (if any) you paid for it by
sending a written explanation to the person you received the work
from. If you received the work on a physical medium, you must
return the medium with your written explanation. The person or
entity that provided you with the defective work may elect to provide
a replacement copy in lieu of a refund. If you received the work
electronically, the person or entity providing it to you may choose to
give you a second opportunity to receive the work electronically in
lieu of a refund. If the second copy is also defective, you may
demand a refund in writing without further opportunities to fix the
problem.

1.F.4. Except for the limited right of replacement or refund set forth
in paragraph 1.F.3, this work is provided to you ‘AS-IS’, WITH NO
OTHER WARRANTIES OF ANY KIND, EXPRESS OR IMPLIED,
INCLUDING BUT NOT LIMITED TO WARRANTIES OF
MERCHANTABILITY OR FITNESS FOR ANY PURPOSE.

1.F.5. Some states do not allow disclaimers of certain implied

warranties or the exclusion or limitation of certain types of damages.
If any disclaimer or limitation set forth in this agreement violates the
law of the state applicable to this agreement, the agreement shall be
interpreted to make the maximum disclaimer or limitation permitted
by the applicable state law. The invalidity or unenforceability of any
provision of this agreement shall not void the remaining provisions.

1.F.6. INDEMNITY - You agree to indemnify and hold the Foundation,

the trademark owner, any agent or employee of the Foundation,
anyone providing copies of Project Gutenberg™ electronic works in
accordance with this agreement, and any volunteers associated with
the production, promotion and distribution of Project Gutenberg™
electronic works, harmless from all liability, costs and expenses,
including legal fees, that arise directly or indirectly from any of the
following which you do or cause to occur: (a) distribution of this or
any Project Gutenberg™ work, (b) alteration, modification, or
additions or deletions to any Project Gutenberg™ work, and (c) any
Defect you cause.

Section 2. Information about the Mission

of Project Gutenberg™
Project Gutenberg™ is synonymous with the free distribution of
electronic works in formats readable by the widest variety of
computers including obsolete, old, middle-aged and new computers.
It exists because of the efforts of hundreds of volunteers and
donations from people in all walks of life.

Volunteers and financial support to provide volunteers with the

assistance they need are critical to reaching Project Gutenberg™’s
goals and ensuring that the Project Gutenberg™ collection will
remain freely available for generations to come. In 2001, the Project
Gutenberg Literary Archive Foundation was created to provide a
secure and permanent future for Project Gutenberg™ and future
generations. To learn more about the Project Gutenberg Literary
Archive Foundation and how your efforts and donations can help,
see Sections 3 and 4 and the Foundation information page at
www.gutenberg.org.

Section 3. Information about the Project

Gutenberg Literary Archive Foundation
The Project Gutenberg Literary Archive Foundation is a non-profit
501(c)(3) educational corporation organized under the laws of the
state of Mississippi and granted tax exempt status by the Internal
Revenue Service. The Foundation’s EIN or federal tax identification
number is 64-6221541. Contributions to the Project Gutenberg
Literary Archive Foundation are tax deductible to the full extent
permitted by U.S. federal laws and your state’s laws.

The Foundation’s business office is located at 809 North 1500 West,

Salt Lake City, UT 84116, (801) 596-1887. Email contact links and up
to date contact information can be found at the Foundation’s website
and official page at www.gutenberg.org/contact

Section 4. Information about Donations to

the Project Gutenberg Literary Archive
Foundation
Project Gutenberg™ depends upon and cannot survive without
widespread public support and donations to carry out its mission of
increasing the number of public domain and licensed works that can
be freely distributed in machine-readable form accessible by the
widest array of equipment including outdated equipment. Many
small donations ($1 to $5,000) are particularly important to
maintaining tax exempt status with the IRS.

The Foundation is committed to complying with the laws regulating

charities and charitable donations in all 50 states of the United
States. Compliance requirements are not uniform and it takes a
considerable effort, much paperwork and many fees to meet and
keep up with these requirements. We do not solicit donations in
locations where we have not received written confirmation of
compliance. To SEND DONATIONS or determine the status of
compliance for any particular state visit www.gutenberg.org/donate.

While we cannot and do not solicit contributions from states where

we have not met the solicitation requirements, we know of no
prohibition against accepting unsolicited donations from donors in
such states who approach us with offers to donate.

International donations are gratefully accepted, but we cannot make

any statements concerning tax treatment of donations received from
outside the United States. U.S. laws alone swamp our small staff.

Please check the Project Gutenberg web pages for current donation
methods and addresses. Donations are accepted in a number of
other ways including checks, online payments and credit card
donations. To donate, please visit: www.gutenberg.org/donate.

Section 5. General Information About

Project Gutenberg™ electronic works
Professor Michael S. Hart was the originator of the Project
Gutenberg™ concept of a library of electronic works that could be
freely shared with anyone. For forty years, he produced and
distributed Project Gutenberg™ eBooks with only a loose network of
volunteer support.
Project Gutenberg™ eBooks are often created from several printed
editions, all of which are confirmed as not protected by copyright in
the U.S. unless a copyright notice is included. Thus, we do not
necessarily keep eBooks in compliance with any particular paper
edition.

Most people start at our website which has the main PG search
facility: www.gutenberg.org.

This website includes information about Project Gutenberg™,

including how to make donations to the Project Gutenberg Literary
Archive Foundation, how to help produce our new eBooks, and how
to subscribe to our email newsletter to hear about new eBooks.
back
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

textbookfull.com