2019 年 28 巻 2 号 （通巻 85 号）

ISSN 1342-2634

日本バイオイメージング学会

バイオイメージング- Program & Abstracts -The 6th International Symposium on Bioimaging
The 28th Annual Meeting of Bioimaging Society

The 27th Annual Meeting of Bioimaging (2018)
Best Imaging Award (Nikon Award)

Polarization-resolved SHG imaging of living tissues
based on multi-point scanning two-photon excitation microscopy

Figure on
cover
Figureonthe
thefront
front
cover

Polarization-resolved SHG imaging of living tissues
based on multi-point scanning two-photon excitation microscopy
Ai Goto,1, 2 Kohei Otomo,1, 2, * Tomomi Nemoto 1, 2
1Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan
2
Graduate School of Information and Technology, Hokkaido University, Sapporo, Japan
E-mail: otomo@es.hokudai.ac.jp
Second harmonic generation (SHG) is a non-linear optical process that can be applied for bioimaging. Twophotons from ultrashort high-peak-power laser pulses are converted to a single photon, and its wavelength becomes
halved via SHG processes. SHG signals are strongly observed from individual non-centrosymmetric molecules,
especially when they are arranged in a crystalline array, such that SHG signals are frequently generated by endogenous
fiber-like structures like supercoiled collagens fibers and thick myosin filaments. Moreover, the probability of SHG
occurring largely depends on the relative orientation between the polarization of the incident light beam and theorientation axis of the targeted molecules. Therefore, polarization-based SHG imaging, the concept of which is based on
analyzing the polarization anisotropy of SHG light or SHG signal intensity as a function of the polarization state of theincident light beam, can be implemented to reveal the structural assembly information of targeted molecules and has
been used as a tool for medical and biological analyses. However, most of polarization-based SHG microscopy requires
a specialized optical setup to irradiate the sample with the excitation laser light beam, capture, and/or analyze the SHG
light signals.
Simple SHG images can be acquired by using a laser scanning microscope equipped with an ultrashorthigh-peak-power laser light source such as a conventional two-photon excitation fluorescence microscope (TPM). Since
most of TPM systems employ a single-point laser scanning method using moving mirrors, their temporal resolution
depends on the speed of the physical movement of the mirrors. However, since early 21st century, multi-point laser
scanning methods have been attempted to achieve high-speed TPM imaging. Recently, a TPM system equipped with aspinning-disk confocal scanning unit (TPM-SD), which incorporates a micro-lens array disk and a Nikpow disk
containing a set of confocal pinholes, has been developed. However, the insufficient energy of conventionally-usedmode-locked titanium-sapphire (Ti-Sa) laser light sources restricted the effective field of view (FOV) of TPM utilizing
the spinning-disk scanning unit (TPM-SD) to a narrow region. Although Shimozawa et al. modified spinning-disks toincrease near infrared laser light power throughput, the system using a Ti-Sa laser light source could use a 10% region of
the effective area of the detector, which corresponded to approximately 40 µm diameter FOV with a 60× objective lens[Shomozawa et al., Proc. Natl. Acad. Sci. USA, 2013]. In order to enlarge the FOV further, we introduced ahigher-peak-power ytterbium (Yb)-based laser light source to the TPM-SD system, achieving approximately 10-times
larger FOV [Otomo et al., Anal. Sci., 2015].
In this study, we introduced a polarization-resolving detection methodology for the TPM-SD system [Goto etal., Front. Phys., 2019], enabling high-speed SHG imaging of living tissue to reveal the structural orientation of collagenfibers. By utilizing developed system, we visualized three-dimensional objects from cross-sectional images of
non-labeled biological specimens. We irradiated a linearly polarized beam to a fixed mouse skin sample from the dermalside and acquired a pair of orthogonally-resolved polarization SHG images from the surface to a depth of 50-µm. As
shown in a figure on the front cover, complicated collagen fiber network structures were visualized, and the signal ratios
between the two channels appeared to reflect their orientations. In addition, we also demonstrated in vivo
polarization-resolved SHG imaging of the collagen fibers in the mouse skeletal muscles at video-rate temporal resolution.
This technique will enable monitoring of the distribution of biological molecular orientations responding to intracellular
dynamics with a superior temporal resolution.

The 6th International Symposium on Bioimaging
The 28th Annual Meeting of the Bioimaging Society

September 21 – 23, 2019
Teikyo University, Tokyo, Japan
Main Organizer
Bioimaging Society
Co-organizers
Teikyo University
National University of Singapore(Mechanobiology Institute, MBI)

Welcome Messages
Dear colleagues,
The Bioimaging Society was esta blished in 1991 in Japan and is the
WelcomeMessages

scientific organizat ion of the bioimaging field. The soc iety co ntributes to t hedevelopment of interdisciplinary r esearch over broad fields suc h as
medic ine, pharmacy, agriculture, physical science, engineering etc. The
Dear
Fromcolleagues,
the Symposium Chairman
research in connection with bioi maging technology such as "Gree nFluorescent Prot ein (GFP) imagi ng", "Super-resolution Microscop y" andThe Bioimaging Society was established in 1991 in Japan and is the scientific organization"Cryo-Electron
Microscopy",
has wonto theNobel Prizein chemistry,andof the bioimaging field.
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and enjoy the Japanese culture and f oods. We will do our very best to makeyou
feel
R y oSuzuki,
S u zwelcome
uPh.D.
k i , P h .Dto. Tokyo and Teikyo University.
Ryo
Chairman
Sincere ly ,C h a i r m aof
n of
The 6th International
SymposiumonBioimaging
The 6 t h I nternational Sy m posium on Bioimaging
The 28th Annual Meeting of the Bioimaging Societyth
28 ofAnnual Meetingt he Bioimaging Societ yProfessor,The
Faculty
Pharma-Science,
Teikyo of
University
P r o f e s s o r , Facu l ty of P ha rma- Science, Teikyo Univ erRyosity Suzuki, Ph .D .
Teikyo U niversity
Chair m a n ofth
I nternational Sy m posium on Bioimaging
The 6
The 28 t h Annual Meeting of t he Bioimaging Societ yTable of Contents
Organization

Organization

X

1

Map
Map		

X

4

GeneralInformation
General Information

X

9

Speakers’ Instruction

X

Speakers’ Instruction
Program

		ProgramSeptember 21, 2019				Saturday 21, September 2019
September
22, 2019 2019
Sunday22, September
Monday23, September
September23, 2019 2019

Abstract

Abstracts

X
X
X

X

Author Index

X

Author Index

20
23
25

X

Acknowledgements

Acknowledgements

11

Annual Generalof Bioimaging
Society
AnnualGeneralMeeting
Meetingof Bioimaging
Society X(In Japanese)

29
114
119
127

(In Japanese)( 総会資料 )The Article of Association of Bioimaging Society
学会定款(In Japanese)X

140

Organization
Main Organizer
Bioimaging Society

Co-organizers
Teikyo University
National University of Singapore (Mechanobiology Institute, MBI)

Partnership
The Society of Chemical Engineers, JapanThe Visualization Society of JapanThe Chemical Society of JapanThe Japanese Society of Microscopy
Japan Society for Cell Biology
The Japan Neuroscience Society
The Japanese Society of Neurochemistry
The Biophysical Society of JapanThe Physiological Society of Japan
Japan Society of Histochemistry and CytochemistryProtein Science Society of JapanThe Japan Society of Ultrasonic in Medicine
The Japan Society of Drug Delivery System
The Physical Society of JapanThe Molecular Biology Society of Japan
Japanese Society for Immunology
The Pharmaceutical Society of Japan
The Academy of Pharmaceutical Science and Technology, JapanThe Japanese Pharmacological Society
Japan Society for Laser Surgery and Medicine

Endorsement
The Japan Society of Applied Physics
Japan Society for Bioscience, Biotechnology, and Agrochemistry─1─SymposiumCommittee
Organizer
Organizing
Chair

Chair

Ryo SUZUKI
Ryo SUZUKI

Teikyo Univ. Fac. Pharma-Sci.
Teikyo Univ., Fac. Pharma-Sci.

President of Bioimaging Society

Presidentof Bioimaging
Society
Takashi
FUNATSUThe
Univ. of Tokyo, Grad. Sch. Pharm.Sci
Takashi FUNATSUThe Univ. of Tokyo, Grad. Sch. Pharm.Sci
Vice President of Bioimaging Society
Vice Presidentof Bioimaging
Society
Etsuko SUZAKI
Shujitsu
Univ., Sch. Pharmacy
Etsuko SUZAKI
Shujitsu Univ., Sch. Pharmacy
Director,
GeneralSociety
Kotaro
OKAAffair of Bioimaging
Keio Univ.,
Fac. Sci. & Tech.
Kotaro OKA
Keio Univ., Fac. Sci. & Tech.

Director, General Affair of Bioimaging Society

Director, Treasurer of Bioimaging Society
Kotaro OKA
Keio Univ., Fac. Sci. & Tech.
Yoshihiro OHTATokyo Univ. of Agriculture Tech., Grad. Sch. Eng.

Director, Treasurer of Bioimaging Society
Members
Yoshihiro OHTA
Kazuo SUZUKI

Members

Tokyo Univ. of Agriculture Tech., Grad. Sch. Eng.
Teikyo Univ. Asia International Institute of Infectious
Diseases Control

Kazuo
SUZUKI
Hiroshi
KIHARA

TeikyoUniv., Asia
International Institute of Infectious
Himeji
Hinomoto
College
Diseases Control (ADC)

Takeharu NAGAI
Hiroshi KIHARAOsaka Univ., The Institute of Scientific and Industrial
Himeji Hinomoto College
Research

Takeharu NAGAI
Kohei SOGA
Kohei SOGA

Osaka Univ., The Institute of Scientific and Industrial Research
Tokyo Univ. of Sci., Fac. Industrial Sci. & Tech.
Tokyo Univ. of Sci., Fac. Industrial Sci. & Tech.

Naoko TANAKA
Naoko TANAKA

Otsuma Women’s Univ., Fac. Home Economics
Otsuma Women’s Univ., Fac. Home Economics

TomomiNEMOTO
TomomiNEMOTO

Hokkaido
Sci.
HokkaidoUniv.,
Univ.,Res.
Res.Institute
Institute for Electronic
Electronic Sci.

International
Organizing
Committee
International Organizing
Committee
PaulMATSUDAIRAMATSUDAIRAPaulNational
Institute(MBI)
NationalUniv.
Univ.ofofSingapore,
Singapore, Mechanobioloyg
Mechanobiology Institute

Latha
SHIVA
LathaKrishnarajpet
Krishnarajpet
SHIVA
NationalUniv.
Univ.ofofSingapore,
Singapore, Mechanobioloyg
Mechanobiology InstituteNational
Institute(MBI)─2─

SymposiumCommittee
Organizer (Continued)
Organizing
Secretary General
Daiki OMATA
Daiki OMATA

Secretary General

Teikyo Univ., Fac. Pharma-Sci.
Teikyo Univ., Fac. Pharma-Sci.

Supporting members (Teikyo University)
Sanae ODA			

Toshiki KUROSAWA

Lisa MUNAKATA

Tadamitsu SHIMA		

Saori KAGEYAMA		

Fumiko HAGIWARA

Yuno SUZUKI		Eri CHIBA			Tamotsu MARUYAMA
Sara ZAKARIA		

Sekihito SAITO		Haruka INOUE

Secretariat
Lab. of Drug and Gene Delivery Res.,
Fac. of Pharm-Sci, Teikyo Univ.
2-11-1 Kaga, Itabashi-ku, Tokyo Japan
int-symp-bioimaging2019@pharm.teikyo-u.ac.jp

─3─

Tomoyuki FUKUZAWA

MapArea Map
Teikyo University
University
2-11-1
Kaga,
Tokyo
173-8605,
JAPAN
2-11-1 Kaga,Itabashi-ku,
Itabashi-ku,
Tokyo
173-8605,
JAPAN

─4─

Map (continued)
AccessAccessTeikyo University
University

─5─Map (continued)

You cancan useuse1F
1Fentrance
entranceatatUniversity
UniversityBldg.
Bldg.

─6─

Map (continued)
rd Floor
University Bld. 3rdRoomviewing
andandExhibition
Room301
301: :Poster
Posterviewing
Exhibition
Room
302
:
Oral
presentation
Room 302 : Oral presentation

─7─

Map (continued)
Room
Room 301
301::Poster
Posterviewing
viewingand
andExhibition
Exhibition

─8─

General Information
Registration
Open
OpenHours
Hours
September
21 21
(Sat)
– 18:30
September(Sat)9:00 9:00
– 18:30
September 22 (Sun) 8:30 – 18:00
September 22 (Sun) 8:30 – 18:00
September 23 (Mon) 8:30 – 15:30
September 23 (Mon) 8:30 15:00
Registration Fees*
Member:Registration 8,000
Fees yen (Active Participant)
3,000
yenyen (Active Participant)Member*:8,000(Graduate Student or Undergraduate Student (Over 5th grade))**3,000 yenFree (Under 4th grade)**(Graduate Student or Undergraduate Student (Over 5th grade))
Freeyen(Under 4th grade)Non-member: 10,000
5,000 yenStudent (Over 5th grade))**Non-member:(Graduate
10,000 Student
yenth or Undergraduate
Free (Under 4 grade)**5,000 yen
(Graduate
Student
or Undergraduate
Student (Over 5th grade))* Member of Bioimaging
Society,
Partnership& Endorsement
Free (Under
4th student
grade) ID at the reception desk** For students, pleaseshow your* Member of Bioimaging Society, Partnership & Endorsement
Banquet (on September 22): 4,000 yen (General), 2,000 yen (Student)** For students, please show your student ID at the reception desk

─9─

General Information (continued)
Symposium
SymposiumInformation
Information
Name
NameBadges
BadgesPleasewear
badge
at allatthe
duringduring
the congressfor identificationand security
Please wearyour
yourname
namebadge
all times
the times
the congressfor identification
andpurpose. Only registered participants wearing a name badge will be allowed access to the session
security purpose. Only registered participants wearing a name badge will be allowed accessrooms.
to the session rooms.
Cloakrd
LocatedCloak in 3 Floor
September
21 (Sat) 9:00 – 18:30
Locatedin 3rd Floor
September 22 (Sun) 8:30 – 18:00
September 21 (Sat)9:00 – 18:30
September 23 (Mon) 9:00 – 15:30
September 22 (Sun) 8:30– 18:00
BanquetSeptember 23 (Mon) 8:30– 15:00
Date and Time : 18:00 September 22
Venue:
Cafeteria “Godereccio”, 1st Floor, University Bldg.
BanquetStyle: Buffet  Dress code: Casual     *Registered applicants only
Date and Time : 18:00 September 22
Venue:
Cafeteria “Godereccio”, 1st Floor, University Bldg.
No
Smoking*Includedisinprohibited
symposium
Smokingin allregistration
areas of Itabashi campus, Teikyo University.
No Photos, No Audio Recording
Photosand audio recording are prohibited.
Electricity
Electric current
CellularPhones is uniformly 100 volts, AC, throughout Japan, but with two different cycles:
50 Hz cellularin easternJapan
including
Kyotoisand
Osaka.Cellular
Leadingphones
hotelsmust
in majorcitiesoffhave
tow
Using
phonesduringthe sessions
prohibited.
be turned
or settosilent moder
duringoutletsof 100and the220sessions.
volts but their sockets usually accept a two-leg plug only.

Electricity
Electric current is uniformly 100 volts, AC, two leg plug, throughout Japan, but with two different
cycles: 50 Hz in eastern Japan including Tokyo and 60 Hz in western Japan including Kyoto andOsaka.

─ 10 ─

Speakers’ Instruction
For oral session

For oral session

All
speakers areto tothethespeaker’s
preparation
area area
15 minutes
prior prior
to your
All speakers
arerequested
requestedtotocome
comespeaker’s
preparation
15 minutes
topresentation.
your presentation.
All the oral presentations are to be made on the PC, and all the speakers are requested to make theirAll
the oral presentations
presentation
data in English.are to be made on the PC, and all the speakers are requested tomake their
datadata
in English.
Pleasesave presentation
your presentation
following the guidelines below, and save on a USB flash drive.
Windows
is theyour
onlypresentation
operating systempresentations.
If youthePlease samedataavailable
followingfor
thetheguidelines
below,
andhave
savepreparedon a USB
presentation
data on a Macintosh,
are advised
to bring
you own
PC. presentations. If you haveflash drive. Windows
is the only you
operatingsystemavailable
for theFor smooth progression of the sessions, speakers are requested to follow the guidelines.
prepared the presentation data on a Macintosh, you are advised to bring you own PC.
・If you have prepared the presentation data on a Macintosh, you are advised to bring you own
Forsmooth progression of the sessions, speakers are requested to follow the guidelines.
computer.
The
If youhave prepared
the presentation
data onMacintosh,
you areto bringyou
6th International
Symposiumon Bioimaging
willa notbe responsible
for advised
any troubles
caused・byown
the computer.
operation or actions that do not follow the guidelines.
 The 6th International Symposium on Bioimaging will not be responsible for any troubles

OScausedand Applicationby the operation or actions that do not follow the guidelines.
OS: Windows 10
Application:
PowerPoint 2016
OS and Application
OS: Windows 10

Monitor(Resolution)
Application:Size
PowerPoint2016
XGA (1024 x 768) *Please check in advance that all the data appear properly under the specified
conditions.
Monitor Size (Resolution)
XGA (1024 x 768) *Please check in advance that all the data appear properly under the
BackupDataspecified conditions.
Please bring backup data to the meeting site if at all possible.

Backup Data

Please bring backup data to the meeting site if at all possible.
Fonts
Please use default-setting fonts of Windows 10.

Font

Please use default-setting fonts of Windows 10.
MoviesFor those who wish to show a movie, it is recommended to bring their own PC to run theMoviespresentation slide.
For those who wish to show a movie, it is recommended to bring their own PC to run thepresentation slide.

─ 11 ─

Speakers’ Instruction (continued)
For Speakers bringing their own PC

For Speakers bringing their own PC Please bring an AC adapter for you PC
bring
an AC
adapter
your PC・PleaseFor PC
with
different
pinfortypes,
please make sure to bring a conversion cable to D-sub
For
PC
with
different
pin
types,
pleasemake sure to bring a conversion cable to D-sub 15 pins・ 15 pins type or HDMI.
   type or HDMI.
 Please prepare backup data either with USB flash drive.
・Please prepare backup data either with USB flash drive.

Chairpersons’Instruction
Chairpersons’ Instruction

All chairpersons
are
asked
to in
betheirin theirsessionroom
later
15 prior
minutesprior
to theAllchairpersons are
asked
to be
sessionroom nolaternothan
15 thanminutesto thebeginning
beginningof the session.
ofthe session.

─ 12 ─

Speakers’ Instruction (continued)
For poster session

For poster session

Posterarearerequested
to follow
thetheschedule
below
in mounting
theirtheirposters
on their
PosterPresenters
Presenters
requested
to follow
schedule
below
in mounting
postersonassignedboards.
Your
posterprogramnumberwillbepostedon
yourassignedboard.
Board
size
their assigned boards. Your poster program number will be posted on your assigned board.
will be 90 cm × 200 cm (width × Height). Pins for mounting will be available. During the poster
Board size will be 90 cm × 200 cm (width × Height). Pins for mounting will be available.
session time, presenters are requested to stand in from of your poster board to be asked questions.
During the poster session time, presenters are requested to stand in from of your poster
board to Mounting/Presentation/Removal
be asked questions.
PosterSchedule

Poster Mounting/Presentation/Removal
Schedulest
Mounting
9:00 – 14:00, September 21 (Sat)
Presentation September 22nd (Sun)stMounting Odd9:00– 14:00,
Septembernumber: 13:00– 13:40 21 (Sat)number : 13:40– 14:20
PresentationEven
September22nd (Sun)rdRemoval
13:00– 15:00,
September
23–
(Mon)
Odd
number:13:00
13:40
Even number: 13:40 – 14:10

PosterRemoval
Summary Oral
Presentation
13:00 – 15:00, September 23rd (Mon)
Preparation:
Pleasefit Summary
the summaryOral
slide Presentation
to slides of ISO A4 size (210 mm × 296 mm) in PowerPoint file.
PosterThesummary slide must be within three slides (including title page). Bioimaging 2019 office
Preparation:
will combine all summary slides in one file. The summary presentation will be performed using
Please fit the summary slide to slides of ISO A4 size (210 mm × 296 mm) in PowerPoint file.
Windows 10 and Microsoft PowerPoint 2016. Please make sure the slide is correctly shown in the
The summaryslide
be within(including
title andpage).
2019include
officeenvironment.
Afterthemustconference,
thethreeofficeslides
will discard
all slides
data.Bioimaging
First slide must
will title,
combineall andsummaryslidesin on file.
The summary
presentation
willsummary.
be performed
using
the
authors
affiliations.
Secondand third
slide can show
your poster
It is possibletoinclude images,
videos andPowerPoint
animations. If
you usevideos,the sure
videosembeddedshownin theWindows
10 and Microsoft
2016.
Pleasemaketheshould
slide beis correctly
slides.
The data size mustlessthan 20 MB.the office will discard all slides and data. First slidein the environment.
Afterbethe
conference,must include the title, authors and affiliations. Second and third slide can show your poster
Presentation:summary.presentation
It is possible
to include
images,videosand animations.
If youuse videos,Summarymust
be within
2 minutes.
If exceed
2 minutes, thepresentation
willthebevideos should
embedded
the slides.will
Thebe
data
size mustbe Windows
less than 10
20 andMB.Microsoft
forcibly
closed.beThesummary inpresentation
performed
usingPowerPoint 2016 in the windows PC of Bioimaging 2019 office.
Presentation:
Summary presentation must be within 2 minutes. If exceed 2 minutes, the presentation will
be forcibly closed. The summary presentation will be performed using Windows 10 andMicrosoft PowerPoint 2016 in the windows PS of Bioimaging 2019 office.

─ 13 ─

Awards
Best Imaging Awards (NIKON Award provided by NIKON INSTECH CO., Ltd., OLYMPUS
Best Imaging Awards (NIKON Award provided by NIKON INSTECH CO., Ltd., OLYMPUS Award
Award provided
by Olympus
Corporation,
HAMAMATSU
Award
provided byHamamatsu
providedby Olympus
Corporation,
HAMAMATSU
Award provided
by Hamamatsu
Photonics
K.K.,
Photonics
K.K., Carl
ZeissbyAward
provided
Carl
ZeissCol., Ltd.)towill
beposterpresented
to fourCarl
Zeiss Award
providedCarl Zeiss
Co., byLtd.)
will
be presented
four
presentersofmemberpresentersof the Bioimaging
Society.
posterof memberof the Bioimaging Society of Japan.
Best Imaging Award (Bioimaging 2019 Award provided byth organization of the 28th Annual Meeting
Imaging Award provided by organization of the 28 Annual Meeting of the Bioimagingof the Bioimaging Society ) will be presented to one person of all poster presenters.
Society will be presented to one person of all poster presenters.
All attendees will vote to select Best Imaging Awards.
All attendees will vote to select Best Imaging Awards.

─ 14 ─Message from Sponsors Providing Best Imaging AwardNIKON Award

OLYMPUS Award

Nikon brings to people the fresh surpriseandinspirations
through
excellentmicroscopic images and conveys theimportance of cutting-edge science andtechnology. We will present the Nikon
Award to researchers and their microscopic
images that will inspire new impressions at
this conference.

OLYMPUS CORPORATION was founded
in 1919, this year we cerebrate 100th
Anniversary. We have supported bio
imaging with many researchers over theyears as the first manufacturer ofcommercializing microscopes in Japan. In
this conference, every year we award thebest imaging prize to support your research.

NIKON INSTECH CO., LTD.

Olympus Corporation

HAMAMATSU Award

Carl Zeiss Award

The letter “イ” was projected on television
about 80 years ago for the first time. The
light technology is evolving rapidly, and wecall the 21st century “the age of light”.
Hamamatsu Photonics, along with light, in
pursuit of the unexplored technology, we willcontinue to develop in the future. We areengaged in research like “grabbing thegoddess’s forehead”.

There are many different ways in whichZEISS promotes science. In 2002, thecompany created the Carl Zeiss ResearchAward to honor outstanding achievementsin the field of bioimaging. ZEISS will bepresenting a research award with a focus on
up-and-coming talent: the Zeiss Award forYoung Researchers.

Hamamatsu Photonics K.K.

Carl Zeiss Co., Ltd.

─ 15 ─

Program

Program at a glance＊These are for members of the Bioimaging Society and related persons.
─ 19 ─

September 21 (Sat)9:55 – 10:00 Opening Remarks
9:55 – 10:00 Opening Remarks
Ryo Suzuki (Chari, Teikyo Univ., Japan)
Ryo Suzuki (Chair, Teikyo Univ., Japan)
10:00 ––12:00
12:00Women
Womenin Science
10:00
in Science
Chairpersons; Etsuko Suzaki (Shujitsu Univ., Japan)
Chairpersons; Etsuko Suzaki (Shujitsu Univ., Japan)
LOK SheeMei (National
Unvi.
of Singapore
Shee-Mei
Lok (National
Unvi. ofSingapore(NUS), (NUS),
Duke-NUS Med. Sci.,Duke-NUS
Singapore) Med. Sci., Singapore)
Speakers;
Kiyoko Kaneko, Hiroko Okinaga (Teikyo Univ. Japan)
S1-1 Teikyo University Support Center Activities for Women Physicians and Researchers
Mako Kamiya (Univ. of Tokyo, Japan)
Kiyoko Kaneko, Yoko Nakayama, Haruko Sekiya, Hiroko Okinaga *LOK Shee(NUS,Physicians
Duke-NUS
Med. Sch.,
Singapore)
Teikyo University Support
CenterMei
for Women
and Researchers,
TeikyoUniversity, Japan
Isabelle Aubert (Univ. of Toronto, Canada)
S1-2

Rapid cancer imaging by rationally designed fluorescence probes
Mako Kamiya,1,* Yasuteru Urano1,2,3,*1
2
12:00 – 13:00
Lunch& Selected
Presentation
Grad. Sch.
Med. andGrad. Sch. Poster
Pharm. Sci.,
The Univ. of Tokyo, 3AMED CREST
Chairperson; Yoshihiro Ohta (Tokyo Univ. of Agriculture Tech., Japan)
S1-3 Capsid
protein structurein Zikavirus
revealsCtr.
the forflavivirus
assembly
process Res., Japan)
Speakers;
Tomoki
Mitani(RIKEN
Biosystems
Dynamics
1,2,7
1,2,7
1,2
Ter Yong Tan , Guntur Fibriansah , Victor A. Kostyuchenko , Thiam-Seng Ng1,2, Xin-Xiang Lim3,
1,2
1,2
5
AkihiroUniv.,
Japan)
Shuijun Zhang1,2, Xin-Ni
LimHasegawa, Jiaqi Wang(Yamaguchi, Jian Shi4, Marc
C. Morais, Davide Corti6, Shee-Mei Lok1,2
1Program in Emerging
Infectious
Diseases,Duke–National
Universityof Singapore
MedicalSchool,ShinyaKomoto(Okinawa
Instituteof Sci.
and Tech.,Japan)

Singapore.
Centre for BioImaging Sciences, Department of Biological Sciences, National University of Singapore,
Singapore.
13:10 – 14:10
The Joint Session with Japanese Society for Molecular Imaging
3
Department of Biological Sciences, National University of Singapore, Singapore.
4
Chairpersons;TomomiUniv.,
Japan)of Singapore, Singapore.
CryoEM unit, Departmentof Nemoto
Biological (Hokkaido
Sciences, National
University
5
Department of Biochemistry
Molecular
Biology,
SealyJapan)
Center for Structural and Molecular
Speakers;
Tomoya andUehara(ChibaUniv.,
Biophysics, University of Texas Medical Branch, Galveston, Texas, United States of America.
6
KenjiroHanaokaof Tokyo,
Japan) Switzerland.
Humabs BioMed SA,
a subsidiaryof Vir (Univ.
Biotechnology,
Inc., Bellinzona,2

S1-4
strategies using
image-guided
focusedultrasoundfor neurodegeneration.
14:30 –Therapeutic
16:00 Bioimaging
Technologies
fromBenchtop
to Clinic
Isabelle Aubert*1,2

Chairpersons;Kohei Soga (Tokyo Univ. of Sci. Japan)
1
Sunnybrook Research Institute, 2University of Toronto, Ontario, Canada.
Yasuaki Kumamoto (Kyoto Pref. Univ. of Med., Japan)
Speakers;
Yasuaki Kumamoto (Kyoto Pref. Univ. of Med., Japan)
12:15 – 13:00 Lunch & Selected
Presentation
NobuakiPoster
Yamanaka(Tokyo Kidney Res. Institute, Japan)
Chairperson; Yoshihiro
Agriculture
Tech., Japan)
Kohei Ohta
Soga(Tokyo(TokyoUniv.
Univ.ofof
Sci., Japan)
P-1

Whole-organ cell profiling accelerated by CUBIC combined with MOVIE imaging

16：15 – 17:00 Plenary Lecture
1,2

Tomoki T. Mitani , Katsuhiko Matsumoto1, Shuhei A. Horiguchi 1, Junichi Kaneshiro 1, Tatsuya C.
3
1
Chairpersons;Kazuo
Suzuki(TeikyoUniv.,
Japan)
Murakami3, Tomoyuki.
Mano, Hiroshi
Fujishima, Ayumu
Konno4, Tomonobu M. Watanabe1, Hirokazu
4
1,3Hirai , Hiroki R. Ueda *Speaker;Paul Matsudaira (National Univ. of Singapore (NUS),
1
RIKEN Ctr. For Biosystems Dynamics Res., 2Osaka Univ. Hosp., 3Grad. Med., Univ. Tokyo,
4
Grad. Med., Gunma Univ. Mechanobiology Institute (MBI, Singapore)

17:15 – 18:15 Poster Summary
─ 20 ─

P-2

Real-time cellular imaging of lung T-lymphocyte accumulation and focus formation in a mouseasthma modelAkihiro Hasegawa,1* Hidetaka Ogino,1 Toshinori Nakayama2
Department of Microbiology and Immunology, Yamaguchi University Graduate School of Medicine,2
Department of Immunology, Graduate School of Medicine, Chiba University1

P-3

From Nano to Macro: Advanced Imaging Techniques for Neuroscience Applications
Shinya Komoto*, Paolo Barzaghi, Koji Koizumi, Toshiaki Mochizuki, Bruno Humbel
Imaging Section, Research Support Division Okinawa Institute of Science and Technology (OIST)

13:15 – 14:15 The Joint Session with Japanese Society for Molecular Imaging
Chairpersons; Tomomi Nemoto (Hokkaido Univ., Japan),Kenjiro Hanaoka (Univ. of Tokyo, Japan)
S2-1

Chemical design of 67/68Ga-labeled antibody fragments for low renal radioactivity levels
Tomoya Uehara
Graduate Sch. Pharma-Sci., Chiba Univ.

S2-2

Development of Far-Red to Near-Infrared Small-Molecule Fluorophores and Their Application
to Fluorescent Probes
Kenjiro Hanaoka 1,*
1
Graduate School of Pharmaceutical Sciences, The University of Tokyo

14:30 – 16:00 Bioimaging Technologies from Benchtop to Clinic
Chairpersons; Kohei Soga (Tokyo Univ. of Sci. Japan)
Yasuaki Kumamoto (Osaka Univ., Japan)
S3-1

Label-free peripheral nerve detection and imaging by Raman scattering
Yasuaki Kumamoto,1,* Tetsuro Takamatsu2
1
Dept. of Pathology and Cell Regulation, Kyoto Pref. Univ. of Medicine,2
Dept. of Medical Photonics, Kyoto Pref. Univ. of Medicine

S3-2

New methodology for examining the light-microscopic glass slide specimen by a Low-vacuum
Scanning Electron Microscope (LVSEM)
Nobuaki Yamanaka
Tokyo Kidney Research Institute

S3-3

Near Infrared Biomedical Imaging for Visualizing Subcutaneous and Submucosal
Information
Kohei Soga1-3, Karina Nigoghossian1, Gil Yeroslavsky2, Thi Kim Dung Doan3,
Masakazu Umezawa1, Kyohei Okubo1,2, Masao Kamimura1,2, and Naoko Ohtani4
1Dept. of Mater. Sci. and Tech., Tokyo Univ. of Sci., Katsushika, Tokyo, Japan
2
Imaging Frontier Center, Tokyo Univ. of Sci., Noda, Chiba, Japan
3
Res. Inst. for Biomed. Sci., Tokyo Univ. of Sci., Noda, Chiba, Japan
4
Dept. of Pathophysiology, Osaka City Univ., Osaka, Japan─ 21 ─

16:15 – 17:00 Plenary Lecture
Chairperson; Kazuo Suzuki (Teikyo Univ., Japan)
PL-1

Imaging the mechanics of early zebrafish development
Jun Zhong1, Dipan Bhattacharya1,3, Sahar Tavakoli1,4, Alexandre Kabla2, Paul Matsudaira1
1
MechanoBiology Institute, Centre for BioImaging Sciences, Department of Biological Sciences, National
University of Singapore, Singapore
2
Department of Engineering, Cambridge University, Cambridge, UK
Current address:
3
Institute FIRC of Molecular Oncology (IFOM), Milan 20139, Italy
4
Harvard University, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge
    02138, USA
Boston Children’s Hospital, Boston 02115, USA
Harvard Stem Cell Institute, Harvard University, Cambridge 02138, USA

17:15 – 18:15 Poster Summary
Summary oral presentation (P-8 ~ P-30)

─ 22 ─

September 22 (Sun)9:00 – 9:45
Poster Summary
9:00 – 9:45 Poster Summary
Summary oral presentation
Summary oral presentation (P-31 ~ P-47)
10:00 – 12:00 Cutting Edge Technologies for Bioimaging
10:00 – 12:00 Cutting Edge Technologies for Bioimaging
Chairpersons; Takeharu Nagai (Osaka Univ., Japan)
Chairpersons; Takeharu
Nagai(OsakaUniv.,
Takeaki
Ozawa(Univ.
of Japan)Tokyo, Japan)
Takeaki Ozawa (Univ. of Tokyo, Japan)
Speakers;
Young-Tae Chang (PSTECH, Korea)
Takeaki
Ozawa(Univ. Imagingof Tokyo,
Japan)
S4-1 Systematic Approachfor Live
Cell Selective
Probe
Development
1,2
Young-Tae Chang Miya Ishihara (National Defense Medical College, Japan)
1
POSTECH Chemistry and 2Center for Self-Assembly & Complexity, IBS
Takeharu Nagai (Osaka Univ., Japan)
S4-2

Imaging and controlling membrane receptor activities in living cells

12:10 – 12:55
Takeaki Lunch
Ozawa1 & Selected Poster Presentation
1
Department of Chemistry, School of Science, University of Tokyo
Chairpersons;Takuya Miyakawa (Univ. of Tokyo, Japan)
Matsumura(TokyoUniv. of Pharm. and Life Sci., Japan)
S4-3 Characterization ofYoshitaka
Photoacoustic
Imaging in
Bioimaging
1
Speakers;
Johtaro Yamamoto (Biomed. Res. Inst., AIST, Japan)
Miya Ishihara,
1
Dept. of Medical Engineering,
National(Kwansei
Defense Medical
College
Mitsuru Yasuda
GakuinUniv., Japan)
YoshitakaMatsumuraS4-4 Trans-Scale Imaging
for Singularity
Biology (Tokyo Univ. of Pharm. and Life Sci., Japan)
Takeharu Nagai1,2 Takuya Miyakawa (Univ. of Tokyo, Japan)
1

The Institute of Scientific and Industrial Research, Osaka Univ.,
Institute for Open and Transdisciplinary Research Initiative., Osaka Univ.

2

13:00 – 14:20 Poster Presentation
Odd number : 13:00 – 13:40
12:15 – 12:55 Lunch
Selected Poster
Even&number: 13:40Presentation– 14:10Chairpersons; Takuya Miyakawa (Univ. of Tokyo, Japan)
YoshitakaMatsumura (Tokyo Univ. of Pharm. and Life Sci., Japan)
14:30 – 15:15 Plenary
LectureChairperson; Takashi Funatsu (Univ. of Tokyo, Japan)
P-4
New evaluation technique for micro-environment using translational and rotational diffusion
Speakers;
Kazuo
Maruyama(Teikyo Univ.,
Japan)
1,
2
2
Johtaro Yamamoto, * Akito Matsui, Masataka Kinjo
1
Biomed. Res. Inst., AIST, 2 Fac. Adv. Life Sci.., Hokkaido Univ.

15:40 – 17:40 The Joint session on Bioimaging between Singapore and Japan
Simultaneous Tracking of Multiple Biomolecules in a Living Filamentous Fungus by Confocal
Chairpersons; Yusuke Toyama (National Univ. of Singapore (NUS),
Raman Microspectroscopic Imaging
MBI, Singapore)
Mitsuru Yasuda,1 Norio Takeshita,2 Shinsuke Shigeto 1,*
1
School of ScienceKaoru
and Technology,
Kwansei Gakuin
University,
Japan
Katoh (National
Instituteof Advanced
Industrial
2
Faculty of Life and Environmental Sciences, University of Tsukuba, Japan
Science and Technology (AIST), Japan)
Speakers;
Toyama(NUS, MBI,monomer
Singapore)
P-6
Attempt
to observeYusuke
the structureof schizophyllan
by Small Angle X-ray Scattering
Tony Kanchanawong(NUS, MBI, Singapore)and Molecular Dynamics
simulation
Yoshitaka Matsumura
and Masaki
Kaoru
KatohKojima(AIST, Japan)
Sch. of Life Sci., Tokyo Univ. of Pharm. and Life Sci.
Kotaro Oka, Yutaka Shindo (Keio Univ., Japan)
P-5

─ 23 ─P-7

Structural basis for the substrate specificity of phosphoenolpyruvate carboxykinases asphosphodiester energy-conversion enzymes
Takuya Miyakawa,1,* Yoko Chiba,2 Yasuhiro Shimane,2 Masaru Tanokura1
1
Grad. Sch. Agric. Life Sci., Univ. Tokyo, 2D-SUGAR, JAMSTEC

13:00 – 14:20 Poster Presentation (P-1 ~ P-47)
Odd number : 13:00 – 13:40
Even number : 13:40 – 14:20

14:30 – 15:15 Plenary Lecture
Chairperson; Takashi Funatsu (Univ. of Tokyo, Japan)
PL-2

Theranostics (Imaging and Therapy) by Lipid Bubbles and UltrasoundKazuo Maruyama
Faculty of Pharma-Science, Teikyo University

15:40 – 17:40 The Joint session on Bioimaging between Singapore and Japan
Chairpersons; Yusuke Toyama (National Univ. of Singapore (NUS), MBI, Singapore)
Kaoru Katoh (National Institute of Advanced Industrial Science andTechnology (AIST), Japan)
S5-1

Mechanical impact of apoptosis in tissue homeostasis
Yusuke Toyama1, 2
1
Mechanobiology Institute, National University of Singapore, Singapore
2
Department of Biological Sciences, National University of Singapore, Singapore

S5-2

Visualizing and Controlling Cell Adhesion Complexes at the Molecular Scale
Tony Kanchanawong 1, 2
Mechanobiology Institute, National University of Singapore, Singapore
2
Department of Biomedical Engineering, National University of Singapore, Singapore

1

S5-3

Fine structures of the cells revealed with conventional and super resolution microscopy
Kaoru Katoh,1,2* Minami Tanaka,1 Totai Mitsuyama 2
1
Biomed. Res. Inst, AIST, 2 AI Res. Center, AIST

S5-4

Mg ion as a novel candidate for second messenger
Kotaro Oka1*, Ryu Yamanaka2, Yutaka Shindo1
1
Dept. Biosci. & Informat., Keio Univ. 2Fac. Pharm., Sanyo-Onoda City Univ.

18:00 – 19:30 Banquet (at Godereccio, Cafeteria, 1F)

─ 24 ─

September 23 (Mon)
9:00 – 10:15 Bioimaging in Drug Delivery Research
9:00 – 10:15 Bioimaging in Drug Delivery Research
Chairpersons; Alexander Klibanov (Univ. of Virginia (UVA), USA)
Chairpersons; Alexander L. Klibanov (Univ. of Virginia, USA)
Yuriko Higuchi (Kyoto Univ, Japan)
Yuriko Higuchi (Kyoto Univ, Japan)
Speakers;
Yu Matsumoto (Tokyo Univ. Japan)
S6-1 Intravital Microscopy
for Drug
DeliveryResearch
Nobuki
Kudo(Hokkaido
Univ., Japan)
Yu Matsumoto
Alexander Klibanov (UVA, USA)

Department of Otolaryngology and Head and Neck Surgery, The University of Tokyo

10:30 –Mechanical
12:00 Bioimaging
ScienceS6-2
interactionsinofBrain
oscillating
microbubbles with cells and a capillary under ultrasoundexposure
Chairpersons; Tomomi Nemoto (Hokkaido Univ., Japan)
Nobuki Kudo
Mutsuo Nuriya (Keio Univ., Japan)
Faculty of Information Science and Technology, Hokkaido University

Speakers;

S6-3

Koichi Homma (Teikyo Univ., Japan)
Targeted Ultrasoundfor Imaging
and(Hokkaido
Drug Delivery
Ryosuke
Enoki
Univ., Japan)222
Siva Dasa , Akshaya
Meher,
PingyuZhang,
ArshadKhan2, Johnny Chen3, Adam Dixon3, Justin Farry3,
Mutsuo Nuriya(Keio Univ.,
Japan)
22
3
1,2,3

Zhongmin Du , Galina Diakova , John Hossack , Alexander L Klibanov *1
Cardiovascular Division, Department of Medicine, 2Cardiovascular Research Center,3
Departmentof Biomedical
Engineering, University of Virginia, Charlottesville USA
12:15 – 13:00
Luncheon
Seminer

Chairperson; Kazuo Maruyama (Teikyo Univ., Japan)
Speakers;
Suguru Kawato (Teikyo Univ., Japan)
10:30 – 12:00 Bioimaging in Brain Science
Tomomi
Nemoto(Hokkaido
13:15 –Chairpersons;
13:45 Young
Research
Award
LectureUniv., Japan)
Mutsuo Nuriya (Keio Univ., Japan)
Chairpersons; Naoko Iida-Tanaka (Otsuma Women’s Univ., Japan)
Honoree;Primes Toshiyuki(TohokuisUniv.,
Japan) Factor for the Critical
S7-1 Imprinting
Learning : Kowada
Thyroid Hormone
the Determining
Period.
Koichi J.Frontiers
Homma in Plant and Food Imaging
14:00 – 15:15
Faculty of Pharmaceutical Sciences, Teikyo University, Tokyo, JAPAN
Chairpersons; Kaszuyuki Kuchitsu (Tokyo Univ., of Sci., Japan)
Masatsugu
Toyota (Saitama
Univ.,
Japan)
S7-2 Visualizing neuronal
circuits controlling
circadian andultradianrhythms in mammals
1,,
Ryosuke
Enoki*
Speakers;
Masatsugu Toyota (Saitama Univ., Japan)
1
Hokkaido University, Research Institute for Electronic Science, Lab of Molecular and Cellular Biophysics
Hirofumi Enomoto (Teikyo Unvi., Japan)
Kazuyuki
Kuchitsu(TokyoUniv., ofJapan)
S7-3 Multimodal multiphoton
imagingof solutes
and solventsin Sci.,the braintissueMutsuo Nuriya 1, 2, 3
Department of Pharmacology, Keio University School of Medicine, 2 Graduate School of Environment and15:15 – 15:25
Awarding Ceremony
Information Sciences, Yokohama National University, 3 JST PRESTO
1

15:25 – 15:30 Closing Remarks
Ryo Suzuki(Chair, Teikyo Univ., Japan)
12:15 – 13:00 Luncheon
Seminar
Chairperson; Kazuo Maruyama (Teikyo Univ., Japan)

─ 25 ─

LS

Brain hippocamp us synthesize s estrogen and androgen which rapidly modulate dendritic
spines in non genomic manner
Suguru Kawato, Mika Soma, Mari Ogiue Ikeda
Dep of Cognitive Neuroscience Sch. Pharma Sci., Teikyo Univ.

13:15 – 13:45 Young Research Award Lecture
Chairpersons; Naoko Iida-Tanaka (Otsuma Women’s Univ., Japan)
AL

Design and synthesis of functional small molecule probes for fluorescence imaging
Toshiyuki Kowada1,2,3
Institute of Multidisciplinary Research for Advanced Materials ,2
Grad uate Sch ool of Science, 3 Grad uate Sch ool of Life Sciences, Tohoku University

1

14:00 – 15:15 Frontiers in Plant and Food Imaging
Chairpersons; Kazuyuki Kuchitsu (Tokyo Univ., of Sci., Japan)
Masatsugu Toyota (Saitama Univ., Japan)
S8-1

Real-time imaging of whole-plant calcium and glutamate dynamics
Masatsugu Toyota,1,2
Department of Biochemistry and Molecular Biology, Saitama University, Japan2
Department of Botany, University of Wisconsin-Madison, USA

1

S8-2

Mass Spectrometry Imaging of Metabolites in Agricultural Products
Hirofumi Enomoto*
Dept. Biosci., Teikyo Univ. Grad. Sch. Sci. Eng., Teikyo Univ. Adv. Inst. Analysis Ctr., Teikyo Univ.

S8-3

Visualizing the Plant Life: Regulation of Development and Stress Responses by the
ROS-Ca2+ Signaling Network and Autophagy
Kazuyuki Kuchitsu1,2, Kenji Hashimoto1,2Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan1

15:15 – 15:25 Awarding Ceremony
15:25 – 15:30 Closing Remarks
Ryo Suzuki (Chair, Teikyo Univ., Japan)

─ 26 ─

Abstract
Abstracts
PL

Plenary Lecture

AL

Young Researcher Award Lecture

LS

Luncheon Seminar

S1

Women in Sciences

S2

The Joint Session with Japanese Society for
Molecular Imaging

S3

Bioimaging Technologies from Benchtop to Clinic

S4

Cutting Edge Technologies for Bioimaging

S5

The Joint Session on Bioimaging between
Singapore and Japan

S6

Bioimaging in Drug Delivery Research

S7

Bioimaging in Brain Science

S8

Frontiers in Plant and Food Imaging

PL1
Imaging the mechanics of early zebrafish development
Jun Zhong1, Dipan Bhattacharya1,3, Sahar Tavakoli1,4, Alexandre Kabla2, Paul Matsudaira1
1.MechanoBiology Institute, Centre for BioImaging Sciences, Department of Biological
Sciences, National University of Singapore, Singapore
2. Department of Engineering, Cambridge University, Cambridge, UK
Current address:
3. Institute FIRC of Molecular Oncology (IFOM), Milan 20139, Italy
4. Harvard University, Department of Stem Cell and Regenerative Biology, Harvard
University, Cambridge 02138, USA
Boston Children’s Hospital, Boston 02115, USA
Harvard Stem Cell Institute, Harvard University, Cambridge 02138, USA
The major morphogenetic movement during gastrulation is convergence and extension when
the spherical symmetry of the blastula is transformed into the bilateral symmetry of the adultbody plan. Gastrulation is powered by the motility, reordering, and shape changes ofindividual embryonic cells under the guidance of chemical morphogen gradients. The
motility of individual cells is mechanically translated into motions of the embryonic tissues
over the entire dorsal and ventral hemispheres. We have mapped the mechanical couplingover the surface of the zebrafish embryo during gastrulation from changes in cell density. The
resulting strain maps identify the mechanical signatures of compression when cells converge
to the dorsal midline, expansion when cells extend to form the head and tail structures, andcompression during somite formation. Interestingly, the two step linear motions ofconvergence and extension can be more simply represented by strain as curl or rotation of thedorsal and ventral hemispheres. What else can be learned from mechanical maps? The strain
maps also indicate stationary points and saddle points which are coincident with key
morphological locations on the embryo surface such as the dorsal organizer and morphogen
gradient source and sinks. This correspondence with the positions of developmentally
important regions of the embryo highlights a possible significance between developmental,
mechanical, and mathematical features.

─ 29 ─PL2
Theranostics (Imaging and Therapy) by Lipid Bubbles and UltrasoundKazuo Maruyama
Faculty of Pharma-Science, Teikyo University
OBJECTIVE“Theranostics” is a treatment strategy that combines therapeutics with diagnostics. The combination of microbubble
and ultrasound (US) is a good tool for “theranostics” due to have multi-potency both of imaging with enhanced echo
signal from bubble and therapeutics with sonoporation of bubble. We have developed a new lipid bubble suitable forsonoporation. For good storage stability and ease of handling, lipid bubbles
were made into freeze-dried formulations. Here we investigate neovascularimaging and enhancement of ERP effect by combination of lipid bubbles andultrasound and describe a new drug delivery technology.
METHODS
Microbubble : Lipid-stabilized bubbles (LB) were prepared by homogenization ofa lipid dispersion in the presence of perfluoropropane gas (Figure 1).
Animal experiments 1: Doxorubicin (DOX) and LB were administered toosteosarcoma-bearing mice via tail vein. Immediately after injection, linear imaging US was exposed to solid tumor
site transdermally to take the neovascular imaging. Then, therapeutic US probe was placed directly on the tumor
surface and therapeutic US was exposed (2MHz, 2W/cm2, Duty cycle, 50 %, burst rate, 2Hz). This treatment was
repeated three times, on days 1, 2 and 4. Animal experiments 2: DOXIL® and LB were co-administered to thyroid
cancer dog and ultrasound treatment (1MHz, 2W/cm2) was done four times.
RESULTS
LBs were in the size range 1-3 µm and could be preserved by freeze-drying and re-constituted by simple addition ofwater. LBs were well suited for imaging of blood flow in tumor. The US theranostics capabilities of LB for the solid
tumor were studied in osteosarcoma-bearing mice. LB was injected to mice via tail vein and linear imaging US was
exposed to solid tumor site transdermally. The flow of LB in blood was observed and neovasuculature of tumor tissue
was imaged clearly. Following, therapeutic US was exposed transdermally over the site of solid tumor tissue.
Oscillation and cavitation of LB induced by low intensity US exposure showed transiently open the tumor blood vessels
and allowing DOX co-injected with LB was delivered into deep area in the tumor tissue. This system achieved anequivalent antitumor effect at about 1/5 the dose in monotherapy of DOX. A thyroid cancer dog was admini-stered
DOXIL® and LB and ultrasonic treatment was
performed four times. This cancer is a good experimentfor proof of concept because it is close to human cancerfrom the viewpoint of spontaneous onset. From the CT
image, the primary tumor volume was clearly reduced(Figure 2). In contrast, because lung metastases were notirradiated with ultrasound, no reduction was observed.
From these results, it was considered that DOXIL® wasefficiently delivered to the tumor tissue from the blood
circulation by LB and ultrasound.
CONCLUSIONS
New freeze-dried LB was stable in vivo for long time.
Oscillation of LB induced by therapeutic US exposure showed transiently open the neovasucular of tumor tissue andallowing DOX or DOXIL® co-injected with LB was delivered into deep area in the tumor tissue. This new approachby the combination of LB and US could delivery medicines and gene into tumor tissue and work better.
REFERENCES
Scale-up production, characterization and toxicity of a freeze-dried lipid-stabilized microbubble formulation for
ultrasound imaging and therapy. J. Unga, S. Kageyama, R. Suzuki, D. Omata, K. Maruyama. J. Liposome Res. 2019─ 30 ─

AL
Design and synthesis of functional small-molecule probes for fluorescence imaging
Toshiyuki Kowada*
Institute of Multidisciplinary Research for Advanced Materials, 2Graduate School of Science,and 3Graduate School of Life Sciences, Tohoku University

1

Fluorescence imaging is one of the most powerful techniques for visualizing biomolecules in living systems, andmany fluorescent sensors and probes have been developed to monitor their activities and concentrations. However, forexample, there is nothing universal, even if you could get imagined what the most useful pH probe is. We still need to
optimize the properties of the probes, such as pKa value of turn-ON fluorescence response, chemical stability,
photostability, and targetability in living organisms. In such a circumstance, we have so far designed and synthesized
fluorescent probes for visualizing cellular functions and ion dynamics by considering structure-optical property
relationship from a physicochemical point of view. In this presentation, I will introduce the research on the developmentof pH-activatable fluorescence probes for intravital imaging of osteoclasts and of fluorescent probes for the quantitative
analysis of zinc ion in intracellular organelles.
Intravital imaging of osteoclasts:1–3 Osteoclasts are multinucleated macrophage-derived cells responsible for bone
resorption in the bone marrow. It is well-known that abnormal bone turnover caused by osteoclast dysfunction strongly
relates to bone diseases, such as osteoporosis and rheumatoid arthritis. The differentiation and function of osteoclasts are
regulated by the surrounding osteoblasts or T cells. Therefore, the development of imaging techniques and chemicalprobes for both cell motility and activity in living animals have been required for understanding the real behavior ofosteoclasts during bone resorption.
We designed and developed pH-activatable fluorescent probes with bone-targeting delivery function and increased
photostability, which enabled long-term monitoring of the bone-resorptive activity of osteoclasts in vivo using
two-photon excitation microscopy. We also demonstrated the quantitation analysis of osteoclast activity. Consequently,
real-time imaging using our probes revealed heterogenic behaviors of osteoclasts in vivo and provided insights into themechanism of bone resorption.
Zinc ion imaging in intracellular organelles: Zinc is an essential trace element abundantly present next to iron in thehuman body. While most part of zinc ion (Zn2+) strongly binds to proteins such as metalloproteins and transcriptional
factors, free Zn2+ is also present at low concentration in organs such as brain and pancreas. In recent years, it has been
pointed out that intracellular free Zn2+ may function as a second messenger in various biological phenomena.
Furthermore, zinc transporters have been shown to regulate the concentration of Zn2+ in individual cellular organelles.
Therefore, the analysis of Zn2+ dynamics in each organelle would be important for understanding Zn2+-mediatedbiological phenomena.
To date, various Zn2+ fluorescent probes based on either small-molecule or protein have been developed, andquantitative analysis of free Zn2+ concentration ([Zn2+]free) in cells has been conducted using fluorescent protein-based
FRET probes. However, the resulting [Zn2+]free values in ER or mitochondria varies between reports probably because of
the difference of the type of probes used or influence from the intraorganellar environment, such as pH variation andoxidative environment. To provide more credible insights, we developed pH-insensitive fluorescent probes forvisualizing free Zn2+ in subcellular compartments and demonstrated quantitative analysis of [Zn2+]free in several
organelles.
1) Kowada, T.; Kikuta, J.; Kubo, A.; Ishii, M.; Maeda, H.; Mizukami, S.; Kikuchi, K. J. Am. Chem. Soc. 2011, 133,
17772–17776.
2) Kikuta, J.; Wada, Y.; Kowada, T.; Wang, Z.; Sun-Wada, G.-H.; Nishiyama, I.; Mizukami, S.; Maiya, N.; Yasuda, H.;
Kumanogoh, A.; Kikuchi, K.; Germain, R. N.; Ishii, M. J. Clin. Invest. 2013, 123, 866–873.
3) Maeda, H.; Kowada, T.; Kikuta, J.; Furuya, M.; Shirazaki, M.; Mizukami, S.; Ishii, M.; Kikuchi, K. Nat. Chem. Biol.
2016, 12, 579–585.

─ 31 ─

LS
Brain hippocampus synthesizes estrogen and androgen which rapidly modulate dendritic
spines in non-genomic manner
Suguru Kawato*, Mika Soma, Mari Ogiue-Ikeda
Dep of Cognitive Neuroscience, Sch. Pharma-Sci., Teikyo Univ.

1

OBJECTIVE
Brain hippocampus locally synthesizes sex steroids (estrogen and androgen) which can modulate synapses in a rapid
non-genomic manner. These processes are essential for memory encoding ability of glutamatergic neurons in the
hippocampus.
METHODS
We analyzed dendritic spines (= postsynapses) in isolated hippocampal slices via visualization of spines withsuper-resolution confocal microscopic imaging. The density of spines and their head diameters were obtained bymathematical and automated software Spiso-3D which identifies spines by calculating geometrical parameters.
RESULTS
Estradiol (E2) at 1 and 10 nM rapidly increased the density of spines, in CA1 glutamatergic neurons. Androgen[testosterone (T) and dihydrotestosterone (DHT)] at 10 nM also increased the density. Using selective kinase inhibitors,
we identified the involvement of individual protein kinases (MAPK, LIMK, PKA, PKC and Src) in spine modulation
signaling through the observation of kinase inhibitor-induced suppression of E2-induced spine increase as well as
suppression of androgen-induced spine increase. Using receptor inhibitors, we identified the involvement of estrogen
receptor (ER) and androgen receptor (AR) in spine modulation signaling through the observation of receptor
inhibitor-induced suppression of E2-induced spine increase as well as suppression of androgen-induced spine increase.
These ER and AR bind spine-membrane via palmitoylation, and they are different from cytosolic/nuclear ER and AR.
CONCLUSIONS
Pathways of spine modulation signaling are as follows: synaptic ERalpha or AR → activation of LIMK, MAPK, Src,
PKA, PKC → phosphorylation of cofilin or cortactin (actin binding proteins) → actin polymerization→ new spine
formation (non-genomic). These pathways are completely different from slow genomic steroid-actions via
cytosolic/nuclear ER and AR.

─ 32 ─

S1-1
Teikyo University Support Center Activities for Women Physicians and Researchers
Kiyoko Kaneko, Yoko Nakayama, Haruko Sekiya, Hiroko Okinaga *Teikyo University Support Center for Women Physicians and Researchers, Teikyo University, JapanBACKGROUND AND OBJECTIVESThe Gender Gap Index is a yearly global ranking of the disparities between the genders published by the World
Economic Forum. Iceland ranked number 1 in 2018, and Japan ranked 110th out of the targeted 149 countries. The
gender gap ranking of Japan is the lowest among member countries of the Organization for Economic Co-operation andDevelopment. While the gender gap of Japan is small in the fields of education and health, the proportion of womenassuming responsibility for roles such as politicians, corporate managers, and managerial staff is extremely low.
The number of female research professionals is increasing every year in Japan, and the relative proportion of female
researchers has also increased by about 15.7%. But compared with other countries around the world, this ratio is rather
low. A questionnaire survey of researchers in academic societies and associations revealed the following reasons for the
low proportion of women researchers: (1) difficulty achieving work-life balance, (2) difficulty returning to work aftermaternity leave, (3) difficult work environments, (4) few role models, (5) gender division of labor, and (6) a culture ofmale privilege.
On this background, we established the Teikyo University Support Center for Women Physicians and Researchers in
April 2013 at our university. In August of the same year, we were entrusted with the “Program for Supporting Research
Activities of Female Researchers” commissioned by MEXT. This is a government-funded program that provides
support for female researchers.
TEIKYO UNIVERSITY SUPPORT CENTER ACTIVITIES FOR WOMEN RESEARCHERS
Our goal is to foster the professional development of women physicians and researchers so they can engage inhigh-quality research activities for the rest of their careers. We also want female physicians and researchers to reach theirfull potential, while still balancing their social roles outside of work.
The 5 basic components in supporting the development of the potential of women are “Environmental Arrangement”,“Improving Education and Research Capabilities”, “Awareness Raising”, “Investigation / Gathering Evidence”, and“Improving the Proportion of Women”.
Under our efforts on “Environmental Arrangement”, we have opened an in-house nursery school as well as an infant
and postnatal childcare facility on the university premises to provide care when a child is ill. We have also introduced the“Concierge Teacher” service, to provide consultations about balancing pregnancy, delivery, child-rearing, and nursing
care. To further assist female researchers during life events and to support the balance between research activities andfamily life, we established the “Researchers’ Support System” where a research assistant supports a female researcher.
Regarding our efforts on “Improvement of Research Abilities”, we organize programs such as seminars on obtaining
external funding, career advancement, and role models. Because of the importance of “Awareness Raising”, we hold anannual symposium on gender equality and workplace reform themes. In response to the President’s statement on genderequality, faculty development seminars on gender equality have been held at several faculties, with discussions on
creating awareness of men and women working together and recognizing the need to promote women.
As a result of these activities, the number of female researchers applying for external funding has doubled and theacceptance rate of women for Grants-in Aid for Scientific Research in 2018 also increased at Teikyo University. In
addition, the total number of women in senior positions (professors and associate professors) has increased, with theproportion rising by 5.2% over the past 6 years.

─ 33 ─

S1-2
Rapid cancer imaging by rationally designed fluorescence probes
Mako Kamiya,1,* Yasuteru Urano1,2,3,*
1
Grad. Sch. Med. and 2Grad. Sch. Pharm. Sci., The Univ. of Tokyo, 3AMED CRESTOBJECTIVE
It has been a long-term goal to develop cancer-imaging techniques that have sufficient specificity and sensitivity, since
early detection and complete resection are an important prognosticator for cancer treatment. Since fluorescence-guided
diagnosis is one of the most powerful techniques for real-time in situ cancer detection, we have focused on developing a
new cancer-imaging method with activatable fluorescent probes targeted to enzymes that are overexpressed in cancer.
METHODS and RESULTS
We first focused on cancer-associated aminopeptidases as imaging targets, since they play essential roles in many
diseases and some of them exhibit altered expression levels in the pathological context. By utilizing our rational design
strategies for activatable fluorescence probes based on the concept of intramolecular spirocyclization, we prepared aseries of fluorogenic substrates for various aminopeptidases which have unique characteristics that the probes themselves
are colorless and almost non-fluorescent due to the formation of spirocyclic structures, but can be converted to highly
fluorescent molecules by the target enzymes, thus showing drastic fluorescence activation. By applying these probes tothe resected specimen from cancer patients, followed by the subsequent assessment of fluorescence signals, we foundthat gGlu-HMRG (a probe for J-glutamyltranspeptidase, GGT) can be selectively activated at certain types of cancers
such as breast cancer, oral cancer, head and neck cancer, and liver cancer. We also found that EP-HMRG targeting
dipeptidylpeptidase-IV (DPP-IV) is effective for detecting esophageal cancer in resected tissue from patients.
In order to expand the target cancer, we next focused on cancer-associated carboxypeptidases. Especially, we focusedon the glutamate carboxypeptidase activity of prostate-specific membrane antigen (PSMA), which is a type IItransmembrane glycoprotein that is overexpressed in prostate cancer. Based on our finding that aryl-glutamate
conjugates with an azoformyl linker are recognized by PSMA and have a sufficiently low LUMO (lowest unoccupied
molecular orbital) energy level to quench the fluorophore through photo-induced electron transfer, we established a new
design strategy for activatable fluorescence probes to visualize carboxypeptidases activity, and developed a first-in-classactivatable fluorescence probe for carboxypeptidases activity of PSMA. We confirmed that the developed probe allowed
us to visualize the CP activity of PSMA in living cells and in clinical specimens from prostate cancer patients, and is
expected to be useful for rapid intraoperative detection and diagnosis of prostate cancer.
CONCLUSIONSIn conclusion, the fluorescence-guided diagnosis by our activatable probes can be used as an imaging guidance during
surgical procedures to help surgeons for efficient tumor inspection. In particular, due to its rapid and strong activation
upon reaction with the target enzyme at cancer site just by topical spraying, we believe that it should have a great
potential for clinical application to realize early detection and complete resection of cancer.

─ 34 ─

S1-3
Capsid protein structure in Zika virus reveals the flavivirus assembly process
Ter Yong Tan , Guntur Fibriansah , Victor A. Kostyuchenko , Thiam-Seng Ng , Xin-Xiang
Lim , Shuijun Zhang , Xin-Ni Lim , Jiaqi Wang , Jian Shi , Marc C. Morais , Davide Corti ,Shee-Mei Lok
1,2,7

3

1,2,7

1,2

1,2

1,2

1,2

4

1,2

5

6

1,2

Affiliations:
Program in Emerging Infectious Diseases, Duke–National University of Singapore Medical
School, Singapore.

1

Centre for BioImaging Sciences, Department of Biological Sciences, National University ofSingapore, Singapore.

2

3

Department of Biological Sciences, National University of Singapore, Singapore.

CryoEM unit, Department of Biological Sciences, National University of Singapore,
Singapore.

4

Department of Biochemistry and Molecular Biology, Sealy Center for Structural andMolecular Biophysics, University of Texas Medical Branch, Galveston, Texas, United Statesof America.

5

6

Humabs BioMed SA, a subsidiary of Vir Biotechnology, Inc., Bellinzona, Switzerland.

Structures of flavivirus (dengue, Zika virus) particles are known to near-atomic
resolution, and show detailed structure and arrangement of their surface proteins (E and, prM
in immature or M in mature virus). In contrast, the arrangement of the capsid proteins:RNAcomplex, which forms the core of the particle, is poorly understood, likely due to inherent
dynamics. Here we stabilized immature Zika virus via an antibody that binds across the E andprM proteins, resulting in a subnanometer resolution structure of capsid proteins within thevirus particle. All secondary structural elements are visible, including a helix that waspreviously thought to be removed via proteolysis. This structure illuminates capsid protein
quaternary organization, including its orientation relative to the lipid membrane and the
genomic RNA, and its interactions with the trans-membrane regions of the surface proteins.
Results show the capsid protein plays a central role in the flavivirus assembly process.
─ 35 ─

S1-4
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─ 36 ─

S2-1
Chemical design of 67/68Ga-labeled antibody fragments for low renal radioactivity levels
Tomoya Uehara
Graduate Sch. Pharma-Sci., Chiba Univ.
OBJECTIVE
Radiolabeled low molecular-weight antibody fragments (LMW Abs) such as Fab exhibit high and persistent
localization of radioactivity in the kidney when they are labeled with metallic radionuclides, which has hindered tumor
visualization near kidney regions and limited therapeutic effectiveness. Persistent renal radioactivity levels were
attributed to the long residence time of radiometabolites generated after lysosomal proteolysis of radiolabeled LMW Abs,
following glomerular filtration and subsequent reabsorption in renal cells. To solve the problem, we hypothesized thatthe renal radioactivity levels after injection of radiolabeled LMW Abs would be improved by liberating a radiometalchelate of urinary excretion from the LMW Abs by enzymes in the lysosome or on the renal brush border membrane(BBM). In this presentation, I will talk the evaluation of the renal radioactivity levels of newly designed 67/68Ga-labeled
antibody fragments with linkages cleaved by enzymes present in the lysosome or on the BBM.
METHODSDi- or tri-peptide, methionine-isoleucine (MI) sequence or methionine-valine-lysine (MVK) sequence were conjugatedto a 67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacylononane-1,4,7-triacetic acid (67Ga-NOTA-SCN) andevaluated as enzyme-cleavable linkages (67Ga-NOTA-MI and 67Ga-NOTA-MVK). In these molecular designs,
67
Ga-NOTA-Met would be liberated from 67Ga-NOTA-MI or 67Ga-NOTA-MVK by the enzymes presented inlysosomes or on the BBM, respectively. Biodistribution of radioactivity after injection of 67Ga-NOTA-MI-Fab,67
Ga-NOTA-MVK-Fab and 67Ga-NOTA-Fab (a conventional thiourea linkage; no enzyme-cleavable linkage) in mice
were evaluated.
RESULTS
67
Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In thebiodistribution study using normal mice, 67Ga-NOTA-MI-Fab showed significantly lower renal radioactivity levels from
1 to 6 h postinjection than those of 67Ga-NOTA-Fab. Analysis of urine obtained 6 h postinjection of 67Ga-NOTA-MI-Fab
in normal mice showed that 67Ga-NOTA-Met was the main radiometabolite. In the biodistribution study using
tumor-bearing mice, 67Ga-NOTA-MI-Fab showed significantly lower renal radioactivity levels without impairing thetumor radioactivity levels at 3 h postinjection than those of 67Ga-NOTA-Fab. However, further studies are required toreduce renal radioactivity levels shortly after the injection of 67Ga-labeled Fabs. To liberate 67Ga-NOTA-Met at an earlier
stage of antibody metabolism in the kidney, 67Ga-NOTA-MVK-Fab that contained BBM enzyme-cleavable linkage wasdesigned. 67Ga-NOTA-MVK-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for
24 h. The MVK sequence conjugated 67Ga-NOTA was recognized by enzymes of renal BBM to liberate
67
Ga-NOTA-Met. 67Ga-NOTA-MVK-Fab showed significantly lower renal radioactivity levels from 30 min to 6 hpostinjection than those of 67Ga-NOTA-Fab and 67Ga-NOTA-MI-Fab. Analysis of urine obtained 6 h postinjection of
67
Ga-NOTA-MVK-Fab in normal mice showed that 67Ga-NOTA-Met was the main radiometabolite. In thebiodistribution study using tumor-bearing mice, 67Ga-NOTA-MVK-Fab showed significantly lower renal radioactivity
levels without impairing the tumor radioactivity levels at 3 h postinjection than those of 67Ga-NOTA-Fab and
67
Ga-NOTA-MI-Fab. In the SPECT/CT images, 67Ga-NOTA-MVK-Fab provided a much higher contrast tumor image
when compared with 67Ga-NOTA-Fab and 67Ga-NOTA-MI-Fab.
CONCLUSIONS
These findings indicated that the strategy of liberating a radiolabeled compound to urinary excretion from antibody
fragments at the renal BBM to reduce the renal radioactivity levels was useful for the 67/68Ga-labeled antibody fragmentsof low renal radioactivity levels.

─ 37 ─S2-2
Development of Far-Red to Near-Infrared Small-Molecule Fluorophores andTheir Application to Fluorescent Probes
Kenjiro Hanaoka 1,*
Graduate School of Pharmaceutical Sciences, The University of Tokyo

1

OBJECTIVE
Fluorescence imaging is one of the most powerful techniques for visualizing temporal and spatial changes of
biological phenomena in living cells, and many fluorescent probes have been developed. In particular, xanthene dyes
such as fluorescein and rhodamines have favorable characteristics, such as high water solubility, high fluorescencequantum yield and high molar extinction coefficient, and they have been utilized as fluorescent cores for fluorescentprobes working in the green to red wavelength region. Recently, silicon-substituted xanthene dye,
2,7-N,N,N’,N’-tetramethyl-9-dimethyl-10-hydro-9-silaanthracene (TMDHS)1), has been reported, and we have also
designed and synthesized Si-rhodamines (SiRs)2,3) and TokyoMagentas (TMs)4), in which the O atom at the 10-positionof xanthene is replaced with a Si atom, as far-red to near-infrared (NIR) fluorescent cores5,6).
In biological systems, the pH in intracellular organelles is strictly regulated, and differences of pH are deeply related
to key biological events such as protein degradation, intracellular trafficking of cell components. To perform thesereactions efficiently, intracellular organelles are maintained at specific pH values appropriate for each process.
Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescent probes havesubstantial limitations, such as inappropriate pKa for imaging in the physiological pH range, inadequate photobleaching
resistance, and insufficiently long excitation and emission wavelengths. So, we set out to develop a novel ratiometric pH
fluorescent probes.
METHODS
Recently, we have established the synthetic scheme for asymmetric SiRs.7) Among them, we discovered the dyescaffold that shows an absorption wavelength change of about 80 nm in response to pH change. Rhodamines are often
used for fluorescence imaging because of their high resistance to photobleaching, high water solubility, and highfluorescence quantum yields. In addition to these advantages, SiRs have longer absorption and emission wavelengths,
which enable multi-color imaging with widely used fluorescent proteins such as GFP and YFP.
RESULTS
We have developed a fluorescent probe, SiRpH5, based on the asymmetric SiR scaffold, which can be used for
imaging of intracellular organelle pH.8) To demonstrate its usefulness for biological applications, we achieved time-lapse
imaging of pH in endocytic compartments during protein trafficking by using transferrin tagged with SiRpH5.
CONCLUSIONS
We have found that asymmetric rhodamines bearing a piperazine ring show a large, pH-dependent blue shift of theabsorption spectra upon protonation of the piperazine ring. SiRpH5 is a fluorophore whose excitation and emission
wavelengths lie in the far-red to NIR region. This provides a new color window for multi-color pH imaging with GFPand YFP. In addition, SiRpH5 has a higher resistance to photobleaching compared to conventional ratiometric probes,
suggesting that SiRpH5 would be a suitable probe for long-term tracking of intracellular organelle pH. Conjugation of
SiRpH5 to macromolecules was an effective strategy for specific imaging of intracellular organelles, and SiRpH5 could
be delivered to the targeted intracellular organelles by choosing an appropriate macromolecule for conjugation. Thesechemical tools should be useful for studying the influence of intracellular pH on biological processes, as well as for invivo imaging.
REFERENCES: 1) Chem. Comm., 1780 (2008); 2) ACS Chem. Biol., 6, 600 (2011); 3) Bioorg. Med. Chem. Lett., 22,3908 (2012); 4) Chem. Commun., 47, 4162 (2011); 5) Review article: Analyst, 140, 685 (2015); 6) Review article: Chem.
Asian J., 12, 1435 (2017); 7) Chem. Commun., 54, 6939 (2018); 8) J. Am. Chem. Soc., 140, 5925-5933 (2018).

─ 38 ─

S3-1
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2322)6:)8-779)7-2%0%&)0*6))%2(232()7869'8-:)1%22)6!359%28-*=8,)(-7'6-1-2%8-32;)'300)'8)(%291&)63*74)'86%3*2)6:)%2(2322)6:)8-779)7)<'-7)(*6316%87-2'09(-2+ 74)'86%*6311=)0-2%8)(%2(921=)0-2%8)(
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%44%6%897 2 8,) %44%6%897 %2 )<'-8%8-32 0%7)6 %8 ?   21 )1-77-32 -7 79&(-:-()( -283  1908-40) &)%17 &= %
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=8,)46-2'-4%0'31432)286)+6)77-32%2(59%(6%8-'(-7'6-1-2%8-32%2%0=7-7;)'0%77-*-)(2)6:)%2(2322)6:)8-779)7
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()8)'8-32;,)6)4096%08%6+)88-779)7%6)83&)-274)'8)(,3;):)6%6%4-(2)6:)-1%+-2+8)',2-59)-76)59-6)(!,-7
6)59-6)1)28;%77%8-7*-)(&=3961908-43-28%44%6%897;)%'59-6)( 74)'86%-2 7)'32(779'')77*900=!,)3:)60%=3*8,)2)6:)46)(-'8-32(-786-&98-32;-8,%&6-+,8*-)0(-1%+)%003;)('3928-2+3*8,)291&)673*2)6:)437-8-:)46)(-'8-32
43-287838,)838%01)%796)1)2843-287%8-2(-:-(9%08%6+)88-779)7=7)88-2+%8,6)7,30(*368,)6%8-33*8,)7)8;3:%09)7
;) 6)%0->)( %8%+0%2') -()28-*-'%8-32 3* 4)6-4,)6%0 2)6:)7 -2 8,) *-)0( 3* :-); *  8%6+)8 8-779)7 -2'09(-2+ 4)6-4,)6%02)6:)7%2( '322)'8-:)8-779)7%2%0=>)(  ;)6)869)46)(-'8-327 "   48!,)7)6)79087-2(-'%8)(8,%88,)1908-43-28%1%274)'8637'34-'8)',2-59);%7'%4%&0)3*6%4-(%2(%''96%8)4)6-4,)6%0
2)6:)-1%+-2+)1%-2-2+-779)73*8,)46)7)28)(8)',2-59)*36-286%34)6%8-:)97)7-2'09()*97-32;-8,%2)2(37'34-'
8)',2-59)%7;)00%7348-1->%8-323*1908-43-28%66%2+)1)28%2()<'-8%8-320%7)6;%:)0)2+8,─ 39 ─

S3-2
New methodology for examining the light-microscopic glass slide
specimen by a Low-vacuum Scanning Electron Microscope (LVSEM)
Nobuaki Yamanaka, Tokyo Kidney Research Institute
OBJECTIVE
Recently an innovative histopathological examination method has been invented. This method
enables the light microscopic glass slide specimen to be directly observed by low-vacuum
scanning electron microscope(LVSEM). To obtain the contrast for EM examination, the lightmicroscopic specimen should be stained by heavy metal. At present, as the heavy metal staining,silver staining and platinum-bleu staining are available for light microscopic paraffin sections
to be observed by LVSEM. This study is to verify the usefulness of this method as correlative
light and electron microscopic (CLEM) analysis in the routine morphological examination.
METHODS
For the renal biopsy diagnosis, silver staining (PASM staining) is routinely implemented, then
PASM-stained glass slide tissue is directly available for observation with LVSEM. For
Platinum-blue staining, usual paraffin section for light microscopy is deparaffinized andimmersed with Platinum-blue solution for 10-20 minutes. These metal-stained sections on theglass slide is observed with Hitachi LVSEM TM3030 for various light microscopic tissue
specimens.
RESULTSIn observation of the specimen with silver staining, the ultrastructural fine details of silverpositive components, such as basement membrane and collagen fibers, are clearly depicted. In
contrast, silver-negative components, such as endothelial cells and podocytes, are finely shownin their cytoplasmic details by the platinum-blue staining. Several new unexpected
ultramicroscopic findings were observed in examining the light microscopic tissue specimens.
CONCLUSIONSThis methodology is highly beneficial for obtaining electron microscopic level informationfrom usual light microscopic specimens with the higher magnification and higher resolution.
The properties of scanning electron microscopy of LVSEM provide additional 3D informationon the entire specimen thickness and further contribute to clear, detailed perception of themorphological changes.
The examination procedures are fairly simple, comparing to the conventional special
technique required for usual EM examination. It is expected this CLEM method will contributeto improve the quality of histopathological analysis of LM specimens.

─ 40 ─

S3-3
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1
Dept. of Mater. Sci. and Tech., Tokyo Univ. of Sci., Katsushika, Tokyo, Japan
2
Imaging Frontier Center, Tokyo Univ. of Sci., Noda, Chiba, Japan
3
Res. Inst. for Biomed. Sci., Tokyo Univ. of Sci., Noda, Chiba, Japan
4
Dept. of Pathophysiology, Osaka City Univ., Osaka, Japan

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─ 41 ─

S4-1
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1
2
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S4-2
Imaging and controlling membrane receptor activities in living cells
Takeaki Ozawa1
1
Department of Chemistry, School of Science, University of TokyoOBJECTIVE
Recognition of extracellular stimuli via various cell-surface receptor proteins is indispensable for cells to adapt tosurrounding dynamic environments. The amount of receptors on plasma membrane is strictly regulated for the precise
modulation of receptor-mediated signaling. To unveil the physiological roles of receptor proteins, direct manipulation of
the receptor-mediated signaling in living cells is required. Conventionally, chemical compounds have been used for the
regulation of receptor activities; however, the diffusion and technical difficulties hampers precise analysis on their
spatiotemporal functions. To control the receptor-mediated signaling in a spatiotemporal manner, we developed an
optogenetic system for inducing light-dependent internalization on a variety of G-protein coupled receptors (GPCRs),
the largest family of membrane receptors, using an endocytosis fragment of GPCR and its binding protein of -arrestin.
METHODS
To induce endocytosis with light illumination, a pair of light-dependently dimerizing plant proteins called
cryptochrome 2 (CRY2) and CIB was used. β-arrestin was attached to CRY2 (ArrestinCRY). E-fragmentCIB was
generated by fusing carboxyl-terminal tail of vasopressin 2 receptor (V2Rct) with CIB. E-fragmentCIB was subsequently
attached to GPCR of interest. Upon light illumination, cytoplasmic arrestinCRY is recruited to E-fragmentCIB located on
plasma membrane by light-dependent CRY2-CIB interaction. Forced interaction between β-arrestin and V2Rct triggers
endocytosis of GPCR labeled with E-fragmentCIB.
RESULTS
To confirm whether light illumination triggers membrane-tethered E-fragmentCIB (Myr-E-fragmentCIB) endocytosis
or not, Myr-E-fragmentCIB was expressed in HEK293 cells and observed under confocal fluorescent microscope. Upon
illumination with blue laser light, endocytic vesicles were clearly observed in cytoplasm. Dyngo4a, a dynamin inhibitor,
treatment abolished the light-induced endocytosis, indicating that the light-dependent Myr-E-fragmentCIB endocytosis
was dependent on clathrin-mediated pathway.
To explore the destination of endocytic vesicles containing Myr-E-fragmentCIB, several organelle were
fluorescently labeled with green fluorescent protein (GFP) during light-induced Myr-E-fragmentCIB endocytosis.
Confocal microscope observation revealed that endocytic vesicles containing Myr-E-fragmentCIB colocalized withearly-endosome after 30 min illumination, then with lysosome after 60 min illumination. The western blot analysis also
revealed that Myr-E-fragmentCIB was subjected to lysosomal degradation upon illumination, suggesting that endocytic
vesicles containing Myr-E-fragmentCIB were delivered to early-endosome and subsequently degraded in lysosome.
Finally, we investigated whether E-fragmentCIB labeling enables GPCR endocytosis by light or not, several GPCRs
labeled with E-fragmentCIB. Blue light illumination on cells expressing Adrenoceptor A2α (ADRA2A) labeled with
E-fragmentCIB (ADRA2A-E-fragmentCIB) triggered endocytosis of ADRA2A-E-fragmentCIB. Quantitative analysis of thefluorescent intensity on plasma membrane revealed statistically significant decrease of ADRA2A-E-fragmentCIB
compared to ADRA2A upon light illumination. Consequently, E-fragmentCIB labeling enabled light-induced endocytosisof target GPCRs.
CONCLUSIONS
We developed an optogenetic system to induce GPCR endocytosis by light using peptide named E-fragmentCIB.
Endocytic vesicles containing Myr-E-fragmentCIB were delivered to early-endosome and then degraded in lysosome.
The amount of cell surface GPCRs labeled with E-fragmentCIB decreased by the light-dependent endocytosis. The
developed system will enable us to directly regulate the receptor-mediated signal transduction in a spatiotemporalmanner by controlling the amount of receptors on plasma membrane by light.
─ 43 ─

S4-3
Characterization of Photoacoustic Imaging in Bioimaging
Miya Ishihara,1Dept. of Medical Engineering, National Defense Medical College

1

The factors influencing bio-optical imaging performance include spatial resolution, maximum depth, andnon-invasiveness. Further, there is a trade-off among these factors, which means that all of these factors are not satisfied
at the same time. A technical challenge in bio-optical imaging is the elimination of this bottleneck.
Photoacoustic (PA) imaging (also called optoacoustic imaging), which visualizes the distribution of optical absorption
molecules, is based on optical excitation and ultrasound detection through a thermoelastic process. PA imaging has theadvantage of being able to use optical spectroscopic characteristics, and this merit can be used for an optical target aswell as fluorescence imaging. PA imaging is different from fluorescence imaging, in that PA signal intensity is inversely
proportional to fluorescence quantum yield, whereas the signal intensity of fluorescence imaging is proportional tofluorescence quantum yield. The larger absorbance results in greater signal intensity. Another feature of PA imaging is
tomography, which has information of depth resolution. Furthermore, because acoustic waves scatter considerably lessthan optical waves in tissue, PA imaging has the advantage of a deeply penetrating imaging.
Our technical solution to the spatial resolution at the deep region is the expansion of the coverage of ultrasonic andoptical focusing technique. We have constructed a PA tomography type device that can observe the whole body of amouse to obtain a deep region PA image (Figure 1), and a PA microscopic type device with optical spatial resolution.
In the talk, I would like to present the various data acquired thus far, and discuss the role of PA imaging in bioimaging.

Figure 1 Photoacoustic tomography for whole body mouse imaging

─ 44 ─

S4-4
Trans-Scale Imaging for Singularity Biology
Takeharu Nagai1,2
1
The Institute of Scientific and Industrial Research, Osaka Univ., 2 Institute for Open and Transdisciplinary
Research Initiative., Osaka Univ.
There exist critical moments, such as the ‘Big Bang’ where “something out of nothing gets created” or in the future,when artificial intelligence might become greater than human intelligence. These points are called singularities. In thefield of biological science, discontinuous critical phenomena are broadly seen, for example, the emergence of life fromthe primordial soup, or the evolution and outbreak of diseases. It has been indicated that only a small number of coreelements are required to bring about discontinuous changes to an entire multi-component system. However, themechanism-of-action that generates such singularity phenomena is not yet certain. In our research project, to look deeply
into singularity cells, we are developing an imaging platform that will achieve both wide field-of-view high-resolution
imaging and high-speed long-term imaging, and corresponding information analysis methods. This will enable us to beat the cutting edge of new scientific fields, where we uncover the underlying mechanisms for the generation of
singularity cells as well as their biological functions.
In order to study the processes that singularity cells, considered as minority entities, bring criticality to an entire system(ex: an organ or whole body), it is necessary to measure, analyze, and examine such biological systems in a holistic
spatial-temporal manner. For this purpose, an imaging system is required for visualization of molecules, cells, andorgans across different length scales. In order to achieve this, we are now developing a unique trans-scale imaging
device that we call AMATERAS (Aspired Multimodal Analytical Tools for Every Rare Activities in Singularity) whichallows us to capture macroscopic spatiotemporal dynamics with microscopic precision: not only the composite trees but
also the whole forest.
In this symposium, I will introduce how the trans-scale imaging device will open the new horizon for singularity
biology.
Acknowledgements
This work is supported by Grant-in-Aid for Scientific Research on Innovative Areas (18H05408), MEXT, Japan─ 45 ─

S5-1
Mechanical impact of apoptosis in tissue homeostasis
Yusuke Toyama1, 2
1
Mechanobiology Institute, National University of Singapore, Singapore
2
Department of Biological Sciences, National University of Singapore, Singapore
Apoptosis, or programmed cell death, is the most common mechanism of eliminating damaged or unnecessary cells
during embryonic development, tissue homeostasis, and certain pathological conditions. When a cell undergoes
apoptosis within a tissue, the apoptotic cell is expelled from its neighboring non- dying cells. It has been shown by manylabs, including ours, that this mechanical process is driven by the formation and contraction of the actomyosin cables inthe dying and the neighboring cells, and/or by the lamellipodial crawling of the neighboring cells. However, how cellmechanics arises upon apoptotic cell extrusion and feedbacks to cellular and molecular function especially in theneighboring non-dying cells is largely illusive. In this presentation, I will present our current understandings of how
mechanical tension and biochemical natures are altered in the neighboring cells as a consequence of apoptosis, and how
these two factors are related to each other.

─ 46 ─

S5-2
Visualizing and Controlling Cell Adhesion Complexes at the Molecular Scale
Tony Kanchanawong 1, 2
1
Mechanobiology Institute, National University of Singapore, Singapore
2
Department of Biomedical Engineering, National University of Singapore, Singapore
Many complex biological functions are performed by supramolecular assemblies self-organized from a diverse
ensemble of proteins. Cell adhesion structures such as the integrin-based focal adhesions and cadherin-basedcell-cell junctions are multi-protein complexes known to transmit, sustain, sense, and respond to mechanical force.
The knowledge of their physical organization is therefore essential for insights into their mechanobiological
functions. Due to the nanometer size scale of the adhesion protein building blocks, the nanoscale is thefunctionally salient length scale for the spatial organization of these molecular complexes. In this talk, I will
discuss our recent studies that sought to 1) decipher the nanoscale architecture of integrin-mediated andcadherin-mediated cell adhesion complexes, using interference-based super-resolution microscopy techniques [1,2]; 2) control force transmission through specific cell adhesion molecules by chemical biology and optogenetic
approaches.
References
1. Liu, J., et al., Talin determines the nanoscale architecture of focal adhesions. Proceedings of the National
Academy of Sciences, 2015. 112(35): p. E4864-E4873.
2. Bertocchi, C., et al., Nanoscale architecture of cadherin-based cell adhesions. Nat Cell Biol, 2017. 19(1): p.
28-37.

─ 47 ─

S5-3
Fine structures of the cells revealed with conventional and super resolution microscopy0LQDPL7DQDND
Kaoru.DRUX.DWRKKatoh,1,2* Minami
Tanaka,1 Totai7RWDL0LWVX\DPD
Mitsuyama 2 (12pt bold)
1
Biomed. Res. Inst, AIST, 2 AI Res. Center, AIST

Growth cones are enlargement of thin cytoplasm at the tips of growing neurites. The growth cones crawl
about and find the path of neuronal elongation. Their movements depend on the dynamics of actin filaments(actin meshwork), which are too small to observe with conventional optical microscopes. We, therefore,
applied polarized light microscope (Polscope), apodised phase contrast microscope, and recently superresolution microscopes (SIM, STED and Spinning disk) to visualize actin cytoskeleton to see actin
cytoskeleton and other structures.
In this presentation, we will summarize the images of fine structures of the cell, which we recorded withconventional (polarized light and apodised phase contrast) and super resolution microscopy. Polscope
revealed radially aligned actin bundles in the growth cones of living unstained cells. Apodised phase contrast
revealed intra nuclear structures and intracellular organelle (mitochondria, ER etc.). Super resolution
microscopes revealed fine structures of actin meshwork. SIM (Structure Illumination Microscope ) visualized
actin meshwork in live cells with resolution of 110 nm as time lapse movie and STED (Stimulated emission
depletion) microscope visualized them in the fixed cells with higher resolution (30-50 nm) and with thin
optical section (140 nm thick in each optical slice). Two camera SIM showed co-localization of actin andactin bundling protein (fascin) in live cells with high resolution and dissociation of fascin from actin bundles
as time lapse records. Spinning disk type super resolution revealed fine structures in the small fish.
Based on the summarized image data, we will discuss future of the imaging with optical microscopes. This
will include big image data (of several TB size (1 million frame images) ), AI and robots in biomedical
imaging with optical microscopy.

─ 48 ─

S5-4
Mg ion as a novel candidate for second messenger
1

Kotaro Oka*, 2 Ryu Yamanaka, 1 Yutaka Shindo1
Dept. Biosci. & Informat., Keio Univ. 2Fac. Pharm., Sanyo-Onoda City Univ.
OBJECTIVE
Mg2+ is recognized as a multi-target analogue regulator that performs many roles, such as stabilizing biologicalmacromolecules, mediators for enzymatic activity, and also circadian timekeeping. Mg2+ has been recognized due to thefollowing physiological properties; (i) it influences wide-ranging biological processes, (ii) its concentration is tightly
controlled within a narrow sub-millimolar range, and (iii) its intracellular dynamics are slow and long-lasting; its
regulatory manner is not all-or-none in contrast to the switch-like signal transduction by the well-established second
messenger Ca2+ 1). Recent studies, however, have reported another role for Mg2+ as a second messenger in immune cells.
These multifaceted characteristics of Mg2+ raise the question of how Mg2+ processes information and how common its
role is as a signaling molecule. We, therefore, investigated the role of Mg2+ as a second messenger in early
developmental stages of neurons with several fluorescent-imaging techniques 2).
METHODSFor visualizing intracellular Mg2+, we have developed KMG-series dyes. In this work, we used KMG104 forvisualizing the cytosolic Mg2+ levels with simultaneously imaging the cytosolic Ca2+ by Fura-red. We focused on theeffects of γ-aminobutyric acid (GABA) and its functional changes during developmental transition. We show that inneurons, GABAA signaling, whose action is not inhibitory but excitatory at early developmental stages of neurons,
triggers Mg2+ release from mitochondria specifically with several fluorescent Mg indicators. By using several
FRET-based sensors for specific phosphorylation, we also demonstrate that released Mg2+ from mitochondria regulates
the ERK, CREB and mTOR signaling pathways. Finally, the Mg2+ function for neural network maturation was also
investigated as the neuronal functional connectivity by Ca2+ imaging and synaptic activity by FM dye imaging.
RESULTSWe found that cytosolic Mg2+ fluctuations within physiological ranges is enough to crucially regulate ERK, CREB, andmTOR activities; quantitative analysis of the Mg2+ concentration-response curves demonstrates that cytosolic Mg2+
regulates the signaling activities of ERK negatively, CREB positively, and mTOR sigmoidally. Together, intracellular
Mg2+ physiologically integrates and coordinates cellular information and Mg2+ is a novel signal transducer for organizing
neural networks.
CONCLUSIONS
We succeed to demonstrate the Mg2+ as a second messenger in neurons. Mg2+ has an important role for facilitating
structural and functional maturation of neural networks.
REFERENCES
1) Yamanaka R, Shindo Y, Oka K. Int. J. Mol. Sci. 20(14): 3439 (2019).
2) Yamanaka R, Shindo Y, Hotta K, Suzuki K, Oka K. Curr Biol. 28(24): 3984-3991 (2018).

─ 49 ─

S6-1
Intravital Microscopy for Drug Delivery Research
Yu Matsumoto
Department of Otolaryngology and Head and Neck Surgery, The University of TokyoOBJECTIVE
To understand and develop the site-specific (targeted) drug delivery, it is essential to observe the extravasation, tissue
penetration and cellular internalization of drug carriers in vivo. In this regard, we have established an intravital confocal
laser scanning microscopy (IVCLSM) technique for spatiotemporal and quantitative in situ analyses of the drug carrier’s
behavior in a living animal.
METHODSIVCLSM utilizes a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The
setup was optimized through various inventions such as custom-designed mouse stage and coverslip holder, exposure
surgery and stabilization under appropriate anesthesia, and tail vein catheterization for timed injection.
RESULTS(1) Size-Dependent Distribution
Extravasation, penetration, and microdistribution of the different sized nanoparticles were compared in hypovascular
human pancreatic BxPC3 tumors. The 30- and 70-nm nanoparticles were labeled with Alexa 488 and Alexa 594
fluorescent probes, respectively, and concurrently injected into tumor-bearing mice. The 30-nm nanoparticles penetrated
into the interstitium, whereas the 70-nm nanoparticles formed perivascular clusters and failed to move toward theinterstitial space. We demonstrated that the enhanced targeting of drugs to cancer cells within tumors by nanoparticles
largely depends on the size.
(2) Nanoparticles Erupts into the Interstitium
To study extravasation and interstitium penetration in detail, we performed longitudinal observation at 10-minuteintervals, which greatly increased time resolution. This led us to image, for the first time, an interesting phenomenon
where tumor vasculature bursts and nanoparticles erupt out into the interstitium. This phenomenon occurred randomly
throughout the study, in scattered and unspecified areas of tumor blood vessels, for durations of 30-60 minutes. Our
discovery should trigger awareness and close investigation of the microdistribution behavior of nanoparticles.
CONCLUSIONSAs described above, IVCLSM can serve as a powerful tool for investigating the in situ behavior of nanoparticles.
IVCLSM can thereby ascertain key barriers in a residing in a living body, facilitating the development of nanoparticles
optimized for sufficient drug delivery.

─ 50 ─

S6-2
Mechanical interactions of oscillating microbubbles with cells and a capillary underultrasound exposure
Nobuki Kudo
Faculty of Information Science and Technology, Hokkaido University
OBJECTIVE
Contrast ultrasonography, a technique in which an ultrasound scanner is used for visualizing microbubbles of several
microns in diameter that have been introduced inside vessels, is now widely used for non-invasive diagnosis. The pressure
range of diagnostic ultrasound pulses reaches several MPas; however, there is almost no volume change in biologicaltissue that mainly consist of incompressible water, causing no mechanical stress by deformation. On the other hand,microbubbles of a contrast agent easily make a large radial oscillation, causing cavitation effects in their surrounding tissue.
Much interest has recently been shown in cavitation effects, especially for applications to ultrasound therapy such as
introduction of a gene or drug into a cell through a perforation created on a plasma membrane (sonoporation), opening of
the blood brain barrier that exists between cerebral endothelial cells (BBB opening), and modulation of neurons deep
inside the brain (neuromodulation). The mechanisms underlying therapeutic applications, however, have still not been
fully elucidated, and we have been studying interactions between a bubble and a cell and a bubble and endothelial cells
inside a capillary under condition of ultrasound exposure. In this presentation, an observation system equipped with aconfocal microscope and a high-speed camera is introduced, and results of recent observations are shown.
METHODS
A confocal unit (C2si, Nikon) and a high-speed video camera (HPV-X2, Shimadzu) were connected to side ports of an
inverted-type microscope (ECLIPSE Ti, Nikon). Human prostate cancer cells (PC-3 cells) were cultured on a soft gel
scaffold, and a cell with an attached microbubble (bubble liposome) of several microns in diameter was observed. A
focused transducer of 1 MHz in resonant frequency was used to generate a one-shot burst pulse of 3 to 50 cycles in durationand 0.2 to 1.0 MPa in peak-negative pressure. The microbubbles and cells were stained using NBD, Cellmask deep red,and Cytox blue, and z-stacked confocal images were captured before and after the ultrasound exposure. Bubble oscillationand interaction with the cell during ultrasound exposure were captured as 256 high-speed frames of 400×250 in pixels,
and the framing rate was set to 10 Mfps, which allows 10 frames to be taken in one cycle of 1-MHz ultrasound.
RESULTSTo clearly visualize the interaction, a cultured cellsample was arranged on an observation chamber toenable observation of the interaction from a lateral
direction. Figure 1 shows the frames selected fromhigh-speed images of an oscillating microbubble
initially adhering to the surface of a cell. The sample Figure 1. Bubble-cell interaction captured by the high-speed camera. Images
was irradiated by a 50-cycle burst pulse of 0.2 MPa in were captured at an expansion phase of (a) initial 2 cycles and (b) the last 2
peak-negative pressure. The 4 frames in Fig. 1(a) show cycles of irradiated ultrasound. The size of the scalebar is 10 µm.
bubble oscillation in the early two cycles and those in Fig. 1(b) show bubble oscillation in later two cycles of the pulse,
indicating that the bubble that initially adhered to the cell surface was introduced inside the cell during bubble oscillation.
CONCLUSIONS
A biological system always maintains its function in the presence of mechanical stress. Our observations of the reaction ofcultured cells to ultrasonically applied mechanical stress are therefore important for elucidating the mechanisms by whichcells change their nature. Our system can apply a force not only in the presence but also in the absence of a microbubble
by changing ultrasound exposure parameters, suggesting the usefulness of the system also for basic studies onmechanotransduction of cells.
─ 51 ─

S6-3
Targeted Ultrasound for Imaging and Drug Delivery
Siva Dasa2, Akshaya Meher2, Pingyu Zhang2, Arshad Khan2, Johnny Chen3, Adam Dixon3,
Justin Farry3, Zhongmin Du2, Galina Diakova2, John Hossack3, Alexander L Klibanov1,2,3*
1

Cardiovascular Division, Department of Medicine, 2Cardiovascular Research Center, 3Department of
Biomedical Engineering, University of Virginia, Charlottesville VA 22908 USA

OBJECTIVE
Ultrasound, unlike other imaging modalities, is capable of not just providing images of biological tissues and organs: it
can also deposit significant amounts of energy to the area of disease, in a focused or unfocused manner. With ultrasound,
we can observe the delivery process in real time, and also trigger release of the sequestered drug from the carrier systems.
Other imaging modalities, such as MRI, optical and photoacoustic imaging, combined with ultrasound, provide
guidance for spatial targeting of therapeutic action, and help assess delivery efficacy.
METHODS
Microbubbles (perfluorobutane gas) were prepared by sonication or amalgamation and stabilized with a lipid monolayerof DSPC and PEG stearate. For targeting to tumor neovasculature, cyclic RGD-PEG-DSPE was added to the bubble
preparation medium, or inserted in the RBC membrane. Biotinylated liposomes were remote-loaded with doxorubicin
driven by ammonium citrate, and attached to biotinylated microbubbles via a streptavidin link. Superheated
nanodroplets (perfluoropentane or perfluorobutane) with a phospholipid shell were prepared by Nuclepore filtration, andattached to RBCs by electrostatic interaction. Doxorubicin was loaded into RBCs by remote loading. Fluorescent dyes,
ICG and calcein, were loaded into RBCs by osmotic shock. Magnetic targeting of RBCs was achieved by placement ofa ½” N52 Neodymium magnet. For tumor targeting and imaging studies, MC38 colon adenocarcinoma cells (agenerous gift of Dr. J. Schlom, NCI) were injected subcutaneously in the hind leg of C57BL/6 mice. Ultrasound wasapplied either with the focused Philips TIPS unit (1 – 1.2 MHz, 1 Hz pulses), or an unfocused Birtcher Megason
physical therapy clinical unit (1 MHz, 3 sec on, 10 sec off intervals), following biweekly intravenous administration oftherapeutic agents. Imaging was performed with Sequoia c512 with 15L8 probe in CPS mode (7 MHz, MI 0.2).
Fluorescence microscopy was applied to monitor doxorubicin delivery to the tissues. Tumor size following therapy wasassessed with a caliper. Photoacoustic studies were performed with a Spectraphysics tunable nanosecond pulse laser.
RESULTS
Delineating tumor mass by ultrasound imaging of tumor vasculature molecular biomarker αvβ3 has been accomplishedwith cRGD-PEG-DSPE microbubbles. Monitoring tumor perfusion is easily achievable by ultrasound imaging; we wereable to observe influx of ultrasound contrast material in a murine tumor model, as well as blood pool ultrasound signal
replenishment following destruction of microbubbles by ultrasound. It was also noticed that high-MI ultrasound tumor
treatment in combination with intravascular microbubbles leads to transient cessation of blood flow in the tumor, which
may be detrimental for tumor drug delivery. Conversion to low-power prolonged pulses from a physical therapy
ultrasound, in combination with doxorubicin-liposome-microbubble complexes resulted in the suppression of tumor
growth. Intravascular perfluoropentane nanodroplets in combination with high pressure focused ultrasound did not resultin the immediate cessation of tumor blood flow; however, tumor growth was still slowed down in relation to controls.
As the next step, we are investigating RBCs, a natural drug delivery vehicle with longer circulation time. We
have developed tools to entrap doxorubicin inside RBCs, up to 0.9 pg per cell. Co-entrapment of magnetic nanoparticles
allows magnetic targeting of drug-loaded RBCs (confirmed by photoacoustic imaging). Molecular targeting with
RGD-decorated RBCs to recombinant αvβ3 Petri dish has been achieved in vitro in a flow chamber study.
Perfluorocarbon nanodroplets were successfully attached to RBC membrane. Fluorescent dyes (ICG and calcein) wereentrapped by osmotic shock and resealing; ultrasound-triggered dye release from nanodroplet-RBCs has been
demonstrated.
CONCLUSIONS.
Ultrasound, in combination with other imaging modalities, offers a set of tools to guide and perform targeted therapeuticinterventions, and assesses the efficacy of therapeutic interventions in real time.
─ 52 ─

S7-1
Imprinting Primes Learning: Thyroid Hormone is the Determining Factor for the Critical Period.
Koichi J. Homma
Faculty of Pharmaceutical Sciences, Teikyo University, Tokyo, JAPANOBJECTIVE
Avian species has highly cognitive functions comparable to those of mammals. In order to reveal the molecularmechanism of early learning in brain such as imprinting behavior, we are using domestic chicks as an experimental
model.
METHODS & RESULTS
Filial imprinting in birds is the process of forming a social attachment during a sensitive or critical period, restricted to
the first few days after hatching. Imprinting is considered to be part of early learning to aid the survival of juveniles by
securing maternal care. Newly hatched chicks follow the first conspicuous moving object and learn the color and shapeof the object and become attached to it. This is the biological mother under normal conditions. However, underexperimental conditions it can be any other object. We are investigating the molecular mechanism of the sensitive periodusing chicks as a model to memorize the plastic bricks for their mother.
We showed that the thyroid hormone determines the start of the sensitive period. Imprinting training causes rapid
inflow of thyroid hormone (T3) into brain, converted from circulating plasma thyroxine. The hormone thus initiates andextends the sensitive period to last more than 1 week via non-genomic mechanisms. Even in non-imprinted chicks
whose sensitive period has ended, exogenous thyroid hormone enables imprinting. In the brain neurons, we showed that
actin polymerization and depolymerization were occurring simultaneously in the dendritic spines during imprinting
using the imaging method of F-actin dynamics. It can also confer “Memory Priming” (MP). Once chicks have achieved
MP, it is maintained for long periods, driving subsequent other learning. MP endowed by imprinting or exogenous T3
clearly enhanced behavioral flexibility in task-switching and reversal learning paradigms in which chicks adjust to theswitch or reversal of task contingencies, while the MP-induced chicks did not show the enhancement under non-switch
or non-reversal condition.
This suggests that avian species may develop highly cognitive functions via MP in order to adapt to environmental
changes. It is possible that the sensitive period for learning closes only if MP is not conferred at an appropriate time ofdevelopment. The closing of the sensitive period may not exist under usual physiological conditions as long as MP is
acquired.
CONCLUSIONS
Our study elucidates the critical role of imprinting to subsequent learning as being governed by the acute action of
thyroid hormone. There may exist determining factors for the sensitive period among higher intelligent animals as well.
The implications of this study reach beyond the sensitive period of imprinting in chicks and deepen our understanding of
the processes of learning.
ReferencesNat. Commun. 3, 1081 (2012), Neuroscience 308, 115-24 (2015), Neurosci. Lett. 612, 32-37 (2016), PLoS One 12(1),e0169643 (2017), Behav. Brain Res. 349, 25-30 (2018), Horm. Behav. 102, 120-128 (2018), Front. Physiol. 9,1837,
eCollection (2018), Front. Physiol. 9, 1740, eCollection (2018)─ 53 ─

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S7-3
Multimodal multiphoton imaging of solutes and solvents in the brain tissue
Mutsuo Nuriya 1, 2, 3
1
Department of Pharmacology, Keio University School of Medicine, 2 Graduate School of Environment andInformation Sciences, Yokohama National University, 3 JST PRESTOOBJECTIVE
Dynamics of bioactive molecules in the tissue is the key determinant of tissue physiology, pathophysiology andpharmacology, which are then regulated by the dynamics of solvent, water, that constitute ~70% of the whole bodyweight. Thus, characterization of solutes and solvents in the tissue is critical in biology and medicine. Importance ofwater dynamics in the tissue has recently been proposed and caught much attention for the brain tissue. Here, it has beensuggested that regulated flow of water in the brain tissue work to clear pathogenic substances and dysfunction of such
dynamics may lead to various diseases such as Alzheimer’s disease.
Despite such importance, investigation of water dynamics in the brain has been difficult due mainly to the lack ofappropriate technique. The major difficulty is to visualize water molecules by microscopy since small size of H2O does
not allow conventional fluorescence-tagging methodology. In this regard, Raman microscopy holds great promises
because Raman scattering can report the existence of molecular vibration and the major source of O-H in the biologicaltissues is water. While conventional Raman imaging suffers from low efficiency and poor tissue penetration, coherent
Raman scattering microscopy using near infrared lasers realizes highly efficient signal detection and deep tissue
penetration. Finally, use of high-power pulsed lasers for coherent Raman scattering simultaneously induces multiphoton
phenomena of other modalities, allowing multifaceted characterizations of biological samples. Thus, we attempted toapply this multimodal multiphoton microscopy technique to the brain tissues to visualize the dynamics of solutes andsolvents.
METHODS
Acute brain slices were prepared from 2 – 3 -week-old C57/BL6 mice and used for analyses. 300 Pm-thick coronalslices of cerebral cortices were prepared and placed in the temperature-controlled chamber under the microscope. The
brain slices were perfused with oxygenated artificial cerebrospinal fluid during the imaging period.
Multimodal multiphoton microscopy system was built based on FV1000MPE Olympus multiphoton system. Emerald
Engine (A.P.E.) was used as the source laser to emit 1,031 nm Stokes beam as well as 515nm frequency doubled beamthat is then fed to optical parametric oscillator to generate ~775 nm Pump laser, both of which were temporally
synchronized and introduced to the scanning system. Resulting coherent anti-Stokes Raman scattering (CARS) signalsof O-H vibration as well as simultaneously emitted fluorescent signals from two-photon excited fluorescent molecules
were collected by the photomultiplier tubes after the narrow band-pass filters.
RESULTSFirst, we tested if we could see O-H vibration signal inside the brain tissue by using CARS microscopy. This revealed
that O-H vibration signal can be readily observed in the middle of the brain slices where the cellular structures andfunctions are best preserved. In addition, cellular fluorescent markers such as sulforhodamine 101 that selectively stain
living astrocytes can be detected simultaneously, allowing characterization of O-H CARS signals in the context ofcellular organizations and structures. Furthermore, live imaging of the CARS signals while exchanging normal
water-based external solution with deuterated water-based one reveal the dynamics of water molecules inside the brain
tissue. Further characterizations of these images under normal and pathological conditions are currently being
undertaken.
CONCLUSIONS
Our preliminary data suggest that multimodal multiphoton microscopy enables simultaneous visualization of solutes
and solvents in the brain tissue. Further applications of this system are expected to contribute to the field of physiology,
pathophysiology and pharmacology of the brain.
─ 55 ─

S8-1
Real-time imaging of whole-plant calcium and glutamate dynamics
Masatsugu Toyota,1,2
1
Department of Biochemistry and Molecular Biology, Saitama University, Japan2
Department of Botany, University of Wisconsin-Madison, USA
OBJECTIVE
Plants, unlike animals, do not have a nervous system, but they can rapidly sense local environmental stresses (i.e., insect
attack and mechanical wounding), transmit this information throughout their body and activate plant-wide defense
responses. However, the molecular machinery underlying such rapid sensory and long-range signal transduction remains
unclear. Here, we show that L-glutamate is a wound signal in plants that leaks from damaged cells/tissues and activates
the GLUTAMATE RECEPTOR LIKE (GLR) family of ion channels in Arabidopsis thaliana, resulting in the intracellular
Ca2+ transmission to distant organs.
METHODS
Using genetically-encoded GFP-based Ca2+ and glutamic acid (Glu) indicators (GCaMP and iGluSnFR, respectively)
and a motorized fluorescence stereo microscope equipped with a sCMOS camera, we have visualized the plant-wide
spatial and temporal dynamics of cytosolic Ca2+ and apoplastic Glu in response to wounding stress in Arabidopsis.
RESULTS
Mechanical wounding caused immediate Ca2+ and Glu increases in the wounded leaf and subsequently this Ca2+ increase
was transmitted via the phloem to distant target leaves (1089 ± 141 μm/s). In these target leaves, defense marker genes
such as OPR3, JAZ5 and JAZ7 and jasmonic acid/jasmonoyl-isoleucine were highly up-regulated within 2 minutes ofmechanical wounding. We also found that the GLR family of Ca2+-permeable channels was required for the
wound-induced Ca2+ transmission and extracellularly-applied Glu in a leaf could trigger the similar systemic Ca2+transmission and resistance responses.
CONCLUSIONS
These data suggest that the plant GLR is a sensor to monitor the apoplastic Glu levels, and when symplastic Glu isleaked out of wounded cells/tissues, GLR rapidly creates the Ca2+ signal propagating throughout the entire plant andactivates systemic resistance responses in distant organs.

─ 56 ─

S8-2
Mass Spectrometry Imaging of Metabolites in Agricultural Products
Hirofumi Enomoto*
Dept. Biosci., Teikyo Univ. Grad. Sch. Sci. Eng., Teikyo Univ. Adv. Inst. Analysis Ctr., Teikyo Univ.
OBJECTIVE
Agricultural products contain various metabolites, such as anthocyanins, flavan-3-ols, sugars, organic acids,
phospholipids, and plant hormones. For example, in strawberries, anthocyanins, flavan-3-ols, sugars, and organic acids
influence the appearance, taste, and antioxidant properties. In pork meat, sphingomyelins (SMs), the major
phospholipids, affect its health-beneficial properties. Plant hormones abscisic acid (ABA) and 12-oxo-phytozienoic acid
(OPDA), regulate the development of immature common bean. However, the spatial distributions of these metabolites
have not been fully elucidated due to the lack of optimal visualization techniques. Mass spectrometry imaging (MSI) is a
powerful technique for visualization of biomolecules in biological tissues. Matrix-assisted laser desorption/ionization(MALDI)-MSI is commonly used for metabolite imaging in plant and animal tissues. Here, we present our studies on
MALDI-MSI to investigate the distribution of various metabolites in several biological samples.
METHODS
Frozen sections were prepared from the biological samples, namely ripe strawberry fruit, pork chop, and immaturecommon bean, using a cryostat (CM 1860, Leica Mycrosystems). The sections were mounted onto indium-tin-oxidecoated glass slides, and a matrix solution, namely 2,5-dihydroxybenzoic acid (DHB) or 1,5-diaminonaphthalene (DAN),
was applied uniformly over the section using an airbrush. ABA and OPDA were derivatized with Girard’s Reagent T(GirT) to enhance their detection intensities prior to matrix application. MALDI-MSI was performed using an
ultrafleXtreme TOF/TOF instrument (Bruker) at positive or negative ion mode. Ion images of metabolites wereconstructed using FlexImaging software (Bruker). To identify metabolites, MALDI-tandem MS was also performed.
RESULTS
Anthocyanins containing pelargonidin- and cyanidin-glycosides, hexose, sucrose, and citric acid were visualized by
DHB in positive ion mode in ripe strawberry fruit. Pelargonidin-glycosides were distributed on the skin, cortical, andpith tissues, whereas cyanidin-glycosides were distributed only on the skin, suggesting that the different distribution
patterns of anthocyanins are dependent on the aglycone moiety rather than the sugar moiety. While hexose wasdistributed almost equally throughout the fruit, sucrose was mainly distributed in the upper side of the cortical and pithtissues. Flavan-3-ol monomers and oligomers, visualized by DAN in negative ion mode, were mainly distributed in thecalyx, vascular bundle, and skin of the ripe strawberry fruit. SMs in pork chops were visualized by DHB containing
potassium in positive ion mode. SMs containing stearic acid were predominantly distributed in the loin and spinalis
muscle, whereas SMs containing palmitic, lignoceric, and nerbonic acids were predominantly distributed in thetransparent tissue, indicating that SM tissue distribution is dependent on the tissue-specific fatty acid composition. ABA
and OPDA were visualized by on-tissue derivatization with GirT and DHB in positive ion mode, in common bean.
ABA-GirT was mainly distributed in the embryo, while OPDA-GirT was localized in the external structures.
CONCLUSIONS
These results suggest that MALDI-MSI is a valuable technique for visualization of various metabolites in agricultural
products. Spatial information of metabolites obtained by MALDI-MSI would be contribute to improve the quality ofstrawberry fruit, assess the health-beneficial properties of each tissue in pork meat, and elucidate the biological role ofplant hormones.
REFERENCES
Enomoto et al., J. Food Sci., 2019, in Press. Doi: 10.1111/1750-3841.14667
Enomoto et al., Rapid Commun. Mass Spectrom., 32, 1565-1572, 2018.
Enomoto et al., J. Agric. Food Chem., 66, 4958-4965, 2018.
─ 57 ─

S8-3
Visualizing the Plant Life: Regulation of Development and Stress Responses by the
ROS-Ca2+ Signaling Network and Autophagy
Kazuyuki Kuchitsu1,2, Kenji Hashimoto1,2
1
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan
Reactive oxygen species (ROS), such as superoxide anion radical (O2•-) and hydrogen peroxide (H2O2), are highly
toxic molecules produced during photosynthesis and aerobic respiration. However, ROS are enzymatically produced ina highly regulated manner spatially and temporally. ROS play key roles in regulating a broad range of physiologicalprocesses, such as growth and development including tip growth of root hairs and pollen tubes, defense responses
against biotic and abiotic stresses as well as cell wall metabolism. Respiratory burst oxidase homologues (Rboh) havebeen identified as ROS-producing NADPH oxidases, which act as key signaling nodes integrating multiple signaltransduction pathways in plants. Rbohs are synergistically activated by Ca2+ binding to the N-terminal EF-hand motifs
and phosphorylation by several families of protein kinases. The ROS-Ca2+ positive feedback regulation seems to beconserved not only in polarized cell growth but also various signaling processes in plants. We have recently identified
and characterized two Rbohs from an emerging model liverwort Marchantia polymorpha. Knockout lines of the two
isozymes established by genome editing revealed that they have distinct physiological roles in development,
morphogenesis and environmental stress responses. We will discuss various imaging techniques to characterize themorphological phenotypes of the rboh mutants such as cell proliferation rate and the cell division pattern during thevegetative development. Evolutionary aspects and molecular regulatory mechanisms of Rboh/ROS-mediated signaling
will also be discussed.
In flowering plants, programmed cell death (PCD) of the tapetum, the innermost layer of the anther, is
one of the most critical and sensitive steps for pollen maturation and fertility. It is severely affected byvarious environmental stresses, which cause serious problems in agriculture. We recently discovered that
autophagy is induced at the uninucleate stage during pollen maturation and required for tapetal PCD,
postmeiotic anther development and nutrient supply to the pollens in rice. We also established an in vivo
imaging system to analyze the dynamics of autophagic flux in rice tapetum cells, and revealed the dynamics
of autophagy during pollen maturation. Possible involvement of Rboh/ROS-mediated signaling andtranscriptional network in the regulation of autophagy and the proper timing of tapetal PCD during antherdevelopment will be discussed.
Numerous pigments plants contain in many organelles such as chloroplasts, cell wall and vacuoles prevent
fluorescence imaging. We are applying various cutting-edge non-invasive imaging techniques for plant research.
Stimulated Raman scattering (SRS) microscopy is a method to obtain the spatial distributions of various constituents bydetecting their characteristic molecular vibrations. High-speed SRS microscope enables rapid image acquisition withmolecular vibrational spectra in the CH-stretching region (2,800 to 3,100 cm-1), and is expected to be widely useful inmedical diagnoses and biological researches. In collaboration with Dr. Yasuyuki Ozeki, we have applied the imaging
system to various plant tissues and cells. Intracellular and extracellular distribution of biological constituents (e.g., fatty
acids, starch granules and phenolic metabolites) was distinctively visualized without any staining processes. We will
discuss prospects for application of the high-speed SRS imaging to plant sciences. Application of laser-induced surface
deformation microscope to analyze the cell surface of plant cells in collaboration with Dr. Toshinori Morisaku and Prof.
Hiroharu Yui will also be discussed.

─ 58 ─

PosterPosterAbstracts
AbstractP-1

Whole-organ cell profiling accelerated by CUBIC combined with MOVIE imaging
Tomoki T. Mitani1,2, Katsuhiko Matsumoto1, Shuhei A. Horiguchi1, Junichi Kaneshiro1, Tatsuya C.
Murakami3, Tomoyuki. Mano3, Hiroshi Fujishima1, Ayumu Konno4, Tomonobu M. Watanabe1,
Hirokazu Hirai4, Hiroki R. Ueda1,3*
1
RIKEN Ctr. For Biosystems Dynamics Res., 2 Osaka Univ. Hosp., 3Grad. Med., Univ. Tokyo,
4
Grad. Med., Gunma Univ.

P-2

Real-time cellular imaging of lung T-lymphocyte accumulation and focus formation in a mouseasthma modelAkihiro Hasegawa,1* Hidetaka Ogino,1 Toshinori Nakayama2
Department of Microbiology and Immunology, Yamaguchi University Graduate School of Medicine,2
Department of Immunology, Graduate School of Medicine, Chiba University1

P-3

From Nano to Macro: Advanced Imaging Techniques for Neuroscience Applications
Shinya Komoto*, Paolo Barzaghi, Koji Koizumi, Toshiaki Mochizuki, Bruno Humbel
Imaging Section, Research Support Division Okinawa Institute of Science and Technology (OIST)P-4

New evaluation technique for micro-environment using translational and rotational diffusion
Johtaro Yamamoto,1,* Akito Matsui,2 Masataka Kinjo 2
1
Biomed. Res. Inst., AIST, 2 Fac. Adv. Life Sci.., Hokkaido Univ.

P-5

Simultaneous Tracking of Multiple Biomolecules in a Living Filamentous Fungus by Confocal
Raman Microspectroscopic Imaging
Mitsuru Yasuda,1 Norio Takeshita,2 Shinsuke Shigeto 1,*
1
School of Science and Technology, Kwansei Gakuin University, Japan
2
Faculty of Life and Environmental Sciences, University of Tsukuba, Japan

P-6

Attempt to observe the structure of schizophyllan monomer by Small Angle X-ray Scatteringand Molecular Dynamics simulation
Yoshitaka Matsumura and Masaki KojimaSch. of Life Sci., Tokyo Univ. of Pharm. and Life Sci.

P-7

Structural basis for the substrate specificity of phosphoenolpyruvate carboxykinases asphosphodiester energy-conversion enzymes
Takuya Miyakawa,1,* Yoko Chiba,2 Yasuhiro Shimane,2 Masaru Tanokura1
1
Grad. Sch. Agric. Life Sci., Univ. Tokyo, 2D-SUGAR, JAMSTEC

P-8

Analysis of myosin II localization during cytokinesis of sea urchin eggs using super-resolution
microscopy
Natsumi Hosoya1,*, Issei Mabuchi2
Faculty of Social Information Studies, Otsuma Women’s University,2
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo

1

P-9

Measurement resolution of diffraction phase microscopy as investigated with reflection
interference microscopy
Shota Ueki,1 Hisashi Hujiwara 1,*
1
Graduate School of Information Sciences, Hiroshima City University

─ 61 ─

P-10

Development of evaluation method of cell morphology using Digital In-line Holographic
Microscope (D-IHM)
Shuhei Yamamoto1, Ryuji Sawada1, Toru Ezure1, Kanna Nagaishi2
Analytical & Measurements Division, Shimadzu Corporation
2
Second Department of Anatomy, Sapporo Medical University

1

P-11

Development of the imaging methods for phenotyping of knockout mice with the X-ray
Computed Tomography
Hirotoshi Shibuya1, Taku Gotoh2, Ruriko Tanabe2, Shintaro Nomura2, Masaru Tamura1,*
1
RIKEN BRC, 2 Nagahama Inst. of Bio-Sci. and Tech.

P-12

Open Source Local Gene Induction System by IR Laser Irradiation
Joe Sakamoto,1, * Yuko Kamikawa,1 Yasuhiro Kamei 1, 2
1Spectrography and Bioimaging Facility, NIBB Core Research Facilities, NIBB,
2
School of Life Science, SOKENDAI

P-13

Microwave-antenna-integrated cell dishes for nanodiamond quantum thermometry
Keisuke Oshimi1, Yushi Nishimura1, Masuaki Tanaka2, Eiji Shikoh 2, Masazumi Fujiwara1, Yoshio Teki1
1
Grad. Sch. Sci. of Osaka City Univ., 2Grad. Sch. Eng. of Osaka City Univ.

P-14

Titanium Dioxide Nanoparticle is a Brilliant Perfusion Contrast Agent for Visualization andDiagnosis of Small-Animal Microvessel.
Ruriko Tanabe1, Taku Gotoh1, Hirotoshi Shibuya2, Masaru Tamura2, Shintaro Nomura1,*
Nagahama Inst. of Bio-Sci. and Tech., 2 RIKEN BRC

1

P-15

Multimodal OTN-NIR/MRI imaging with IR-1061@Gd-DOTA imaging molecules
Doan Thi Kim Dung1,4, Masakazu Umezawa2, Karina Nigoghossian2, Gil Yeroslavsky3, Kyohei Okubo2,3,Masao Kamimura2,3, Masayuki Yamaguchi4, Hirofumi Fujii4, and Kohei Soga2,3
1
Research Institute of Biomedical Science, Tokyo University of Science,2
Department of Material Science and Technology, Tokyo University of Science,
3
Imaging Frontier Center (IFC), Research Institute for Science and Technology (RIST), Tokyo Universityof Science,4
Exploratory Oncology research & Clinical Trial center, National Cancer Center Hospital East, Japan.

P-16

Development of Functional 19F MRI Contrast Agents Using Perfluorocarbon-Encapsulated
Nanoparticle
Yuki Konishi1, Kazuki Akazawa1, Rena Akimoto1, Fuminori Sugihara2,3, Masafumi Minoshima1, andKazuya Kiuchi1,21Graduate School of Engineering, Osaka University, Osaka, Japan
2
Immunology Frontier Research Center, Osaka University, Osaka, Japan
3
Research Institute for Microbial Diseases, Osaka University, Osaka, Japan

P-17

Visualization of mitochondrial Ca2+ flux in pancreatic INS-1 cells cultured by the medium
with high concentration of fatty acids.
Mariko Suzuki,1 Megumi Kawakubo,1 Naoko Iida-Tanaka2,*
1
Dept. of Health and Nutr. Sci., Grad. Sch. of Stud. in Hum. Cult., Otsuma Women’s Univ.,
2
Dept. of Food Sci., Otsuma Women’s Univ.

P-18

Anti-apoptotic properties of ferulic acid in pancreatic INS-1 cells.
Sumika Takahashi, Megumi Kawakubo, Naoko Iida-Tanaka*
Dept. of Food Sci., Otsuma Women’s Univ.
─ 62 ─

P-19

Cell Morphology and Early-phase Ca2+ Transients of Guinea-Pig Pulmonary Vein
Cardiomyocytes Compared with Atrial and Ventricular Cardiomyocytes
Iyuki Namekata,1 Haruhito Hiiro,1 Tamano Ohmori,1 Yusuke Tanaka,1 Shogo Hamaguchi,1 Hikaru Tanaka1
Department of Pharmacology, Toho University Faculty of Pharmaceutical Sciences

1

P-20

Imaging stress-responsive long-distance ROS-Ca2+ signaling in Marchantia
Kenshiro Watanabe,1 Kota Hasegawa1, Hiroki Shindo1, Kenji Hashimoto1,2, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan

P-21

Regulation of cell proliferation and differentiation by NADPH oxidase-mediated ROS
production in the development of a model liverwort Marchantia polymorphaKazuyuki Kuchitsu1,2, Yuki Hagiwara1, Daisuke Miyamoto1, Kenji Hashimoto1,2Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan

1

P-22

Imaging of ROS dynamics and roles of the ROS-producing NADPH oxidase in tapetal
programmed cell death during pollen maturation in rice
Togo Fukunaga1, Jumpei Sawada1, Kazunori Ogawa1, Bunki Toh1, Kenji Hashimoto1,2, Shigeru Hanamata2,3,
Seijiro Ono4, Ken-ichi Nonomura4, Takamitsu Kurusu2.5, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo University of Science,3
Grad. Sch. Sci. & Tech., Niigata Univ., 4Natl. Inst. Genetics, 5Fac. Eng., Suwa Univ. of Science

P-23

Imaging autophagy in the anther tapetum cells during pollen maturation in rice
Kazunori Ogawa1, Jumpei Sawada1, Togo Fukunaga1, Bunki Toh1, Kenji Hashimoto1,2, Shigeru Hanamata2,3,
Seijiro Ono4, Kenichi Nonomura4, Takamitsu Kurusu2,5, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo University of Science,3
Grad. Sch. Sci. & Tech., Niigata Univ., 4Natl. Inst. Genetics, 5Fac. Eng., Suwa Univ. of Science

P-24

Dynamic Photocontrol of Biomachines During Cell Division
Kazuya Matsuo1, Mafy Nusrat Noushaba1, Shota Hiruma2, Ryota Uehara2, Nobuyuki Tamaoki1
1Research Institute for Electronic Science, Hokkaido university
2
Graduate School of Life Science, Hokkaido University

P-25

Development of Ratiometric Oxygen Probes Based on Green Fluorophore and Red Phosphor
for Visualization of Intracellular Oxygen Level
Mami Yasukagawa, Seiji Tobita, Toshitada Yoshihara*
Department of Chemistry and Chemical Biology, Graduate School of Science and Technology,
Gunma University

P-26

Cell cycle dependence of phototoxicity caused by irradiation of blue laser light
Naohiro Kanamaru1*, Mikiya Yamaguchi1, Tetsuya Matsuyama1, Kenji Wada1, Koichi Okamoto1,
Ai Kawakita2, Kaori Murata2, Kenji Sugimoto2
1
Graduate School of Engineering, Osaka Prefecture University
2
Graduate School of Life and Environmental Sciences, Osaka Prefecture University

P-27

Development of novel microbubbles for ultrasound imaging
Tamotsu Maruyama1, Daiki Omata1, Lisa Munakata1, Tadamitsu Shima1, Saori Kageyama1,
Fumiko Hagiwara1, Yuno Suzuki1, Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma Science, Teikyo University

─ 63 ─

P-28

Contribution of cellular immune response in tumor growth suppression by the combination of
microbubbles and ultrasoundEri Chiba1, Lisa Munakata1, Daiki Omata1, Tadamitsu Shima1, Saori Kageyama1, Fumiko Hagiwara1,
Yuno Suzuki1, Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma Science, Teikyo University

P-29

Evaluation of Transporter Function in Brain Microvascular Endothelial Cells Derived from
Human Induced Pluripotent Stem Cells
Toshiki Kurosawa,1Yuma Tega,1 Tomoko Yamaguchi,3 Tatsuki Mochizuki,2 Yasuhiro Hayashi,1Atsushi Yamashita,1 Hiroyuki Kusuhara,2 Kenji Kawabata,3 Yoshiharu Deguchi1,*1Fac. Pharm-Sci., Teikyo Univ., 2 Grad.Sch. Pharma-Sci., Univ. Tokyo.,
3
Nat. Instit. Biomed. Innov. Health Nut.

P-30

Development of new anticancer drug delivery system with ultrasound for cancer therapy
Yuno Suzuki1, Daiki Omata1, Lisa Munakata1, Tadamitsu Shima1, Saori Kageyama1, Fumiko Hagiwara1,
Kazuo Maruyama2, Ryo Suzuki 1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma-Science, Teikyo University

P-31

Induction of the accelerated blood clearance (ABC) phenomenon by polyethyrene glycol (PEG)
modified microbubbles
Tadamitsu Shima1, Tamotsu Maruyama1, Takumi Yamada1, Daiki Omata1, Lisa Munakata1,
Saori Kageyama1, Fumiko Hagiwara1, Yuno Suzuki1, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University

P-32

Development and evaluation of antibody-modified nanobubbles using Fc binding polypeptides
for tumor imaging
Nobuhito Hamano1, Yusuke Yano1, Yamato Kikkawa2, Tomomi Kobayashi1, Yoko Endo-Takahashi1,
Ryo Suzuki3, Kazuo Maruyama3, Motoyoshi Nomizu2, and Yoichi Negishi1*
1
Dept. of Drug Deliv. and Mol. Biopharm., School of Pharmacy, Tokyo Univ. of Pharmacy and Life Sci.,
2
Dept. of Clin. Biochem., School of Pharmacy, Tokyo Univ. of Pharmacy and Life Sci.,
3
Lab. of Drug and Gene Deliv. Res., Faculty of Pharma-Sci., Teikyo Univ.

P-33

CpG oligodeoxynucleotide loaded lipid nanoparticles activate CD8+ T cells and exert antitumor
effect for murine colon carcinoma
Lisa Munakata1, Daiki Omata1, Yasunari Haseda2, Shohei Koyama3, Yoshiaki Okada4, Yasuo Yoshioka2, 4,
Taiki Aoshi2, Ryo Suzuki1, *1Faculty of Pharma-Science, Teikyo University, 2 Research Institute for Microbial Diseases, Osaka
University,
3
Graduate School of Medicine, Osaka University
4
Graduate School of Pharmaceutical Sciences, Osaka University

P-34

Development of fluorescence / NanoSIMS bioimaging coupled with immunostaining usingnovel hybrid probe
Naoya Iwano,1 Wataru Haya,1 Yasuhiko Fujita,1 Kohji Shimoda, 2 Yasushi Miyauchi,3 Takuo Onizuka1
Toray Research Center, Inc., 2 Toray Industries, Inc., 3 Kamakura Techno-Science, Inc.

1

─ 64 ─

P-35

Promotion of α2,3-Sialylation of Glycoproteins by a Passport Sequence Tag Recognized by
Cargo Receptor
Rino Yamada1, Hirokazu Yagi1, Rena Honda2,3,5, Maho Yagi-Utsumi1,2,3,5, Kazuhiro Aoki2,4,  
Koichi Kato1,2,3,5, *1
Graduate School of Pharmaceutical Sciences, Nagoya City University,
2
Exploratory Research Center on Life and Living Systems (ExCELLS),
3
Institute for Molecular Science,  4National Institute for Basic Biology, National Institutes of Natural Sciences,
5
School of Physical Science, SOKENDAI (The Graduate University for Advanced Studies)

P-36

Quantitative analysis of cell cycle progression within heterogeneous cell population using
information theory
Ryu Yamanaka,1, 2 Kotaro Oka1, 3, 4, *1
Dep. of Biosci. and Info., Fac. of Sci. and Tech., Keio Univ., Japan,
2
Faculty of Pharm. Sci., Sanyo-Onoda City Univ., Japan,
3
Grad. Inst. of Med., Col. of Med., Kaohsiung Med. Univ., Taiwan,
4
Waseda Res. Inst. for Sci. and Eng., Waseda Univ., Japan. *Corr. Auth.

P-37

Simultaneous visualization of intracellular energy substrates by using dual FRET imaging
Yutaka Shindo,1 Mitsuru Iizumi,1 Ryu Yamanaka,2 Kohji Hotta,1 Kotaro Oka1,3,4,*
1
Biosci Info., Keio Univ., 2 Pharmac. Sci. Sanyo-Onoda City Univ., 3 Inst. Med., Kaohsiung Med. Univ.,
4
Waseda Res Inst. Sci., Waseda Univ.

P-38

Intra-vital fluorescence imaging of interaction between transplanted stem cells and immune
cells by quantum nano-materials and tissue clearing (CUBIC) technology
Toshiki Mizumaki,1,* Hiroshi Yukawa,1,2,3 Kodai Kitamura,1 Daisuke Onoshima, 1,2 Etsuo A. Susaki, 4,5
Hiroki R. Ueda, 4,5 Yoshinobu Baba, 1,2,3,6
1
Dept. Biomolecular Eng., Grad. Sch. Eng., Nagoya Univ.,
2Inst. Nano-Life-Systems, Inst. Innovation for Future Society, Nagoya Univ.,3
National Institute of Radiological Science (NIRS), National Institute for Quantum and Radiological   
Science and Technology (QST),
4
Dept. Systems Pharmacology, Grad. Sch. Med., Tokyo Univ.,
5
Laboratory for Synthetic Biology, Center for Biosystems Dynamics Research, RIKEN,
6
Health Research  Institute, AIST

P-39

Brain-targeted Gene Delivery using Microbubbles and UltrasoundDaiki Omata1, Fumiko Hagiwara1, Lisa Munakata1, Tadamitsu Shima1, Saori Kageyama1, Yuno Suzuki1,
Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma-Science, Teikyo University

P-40

Blood-brain barrier opening using 220-kHz transcranial MRI-guided focused ultrasound andmicrobubbles in mouse and rat
Michiharu Yoshida1, Kazuo Maruyama2*, Yasutaka Kato3, Rachmilevitch Itay4, Shuji Suzuki4,
Omer Brockman4, Yeruham Shapira4, Kenji Moriyama5, Daiki Omata2, Ryo Suzuki2, Hajime Kamada1
1
Department of Neurosurgery, Hokuto Hospital,
2
Faculty of Pharma-Sci., Teikyo Univ.,
3
Laboratory of Cancer Med-Sci., Hokuto Hospital,
4
InSightec Ltd.,
5
Department of Radiology, Hokuto Hospital─ 65 ─

P-41

Enhanced chemotherapy effects on gynecological cancers by using the combination of lipid
bubble and ultrasoundKohei Yamaguchi1, Yoko Matsumoto1 *, Haruka Nishida1, Daiki Omata2, Michihiro Tanikawa1,
Kenbun Sone1, Katsutoshi Oda1, Yutaka Osuga1, Ryo Suzuki2, Kazuo Maruyama2, Tomoyuki Fujii1
1
The Department of Obstetrics and Gynecology, The University of Tokyo
2
Laboratory of Drug and Gene Delivery System, Faculty of Pharma-Science , Teikyo University

P-42

Bioimaging of inhalable nanocarriers with aggregation-caused quenching dye to visualize
retention in the respiratory system
Kohei Yamada,1 Tetsuya Matsushita,1 Kurt D. Ristroph, 2 Hoang D. Lu, 2 Yoshiki Seto, 1 Wei Wu, 3
Hak-Kim Chan, 4 Robert K. Prud’homme, 4 Hideyuki Sato, 1 Satomi Onoue 1,*
1
Laboratory of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka, Japan,
2
Department of Chemical & Biological Engineering, Princeton University, USA,
3
Key Laboratory of Smart Drug Delivery of MOE & PLA, Fudan University, China
4
Advanced Drug Delivery Group, The University of Sydney, Australia

P-43

Development of Caged Gene Regulators for Application to Control of Genetic Recombinationin vivo
Kurumi Yoshida,1,* Masafumi Minoshima,1 Kazuya Kikuchi 1,2
Graduate School of Engineering, Osaka University. 2 Immunology Frontier Research Center, Osaka Univ.

1

P-44

Image evaluation of biological tissue using Bio-LSI
Ryoma Kumagai,1 Ankush Prasad,2 Teruya Sato,1 Kosuke Ino,3 Hitoshi Shiku,3Shigenobu Kasai1
Graduate Department of Electronic, Tohoku Institute of Technology, Sendai, Japan,
2
Department of Biophysics, Centre of the Region Haná for Biotechnological and Agricultural Research,
Faculty of Science, Palacký University, Olomouc, Czech Republic
3
Graduate School Department of Applied chemical – electrochemical science and technology, Tohoku
University, Sendai, Japan

1

P-45

Study on Photodynamic diagnosis for micrometastases using photosensitizer Talaporfin sodium
Saito Takuma1 , Furuya Kazunari1 , Li Liming1
1
Graduate School of Chitose Institute of Science and Technology

P-46

In vivo imaging of exosomes derived from stem cells by using quantum nano-optics forregenerative medicine
Yuka Kumon,1 Hiroshi Yukawa,1,2,3 Onoshima Daisuke 1,2, Yoshinobu Baba1,2,3,4
Grad. Sch. of Engineering, Nagoya Univ., 2 Institutes of Innovation for Future Society, Nagoya Univ.
3
National Institutes for Quantum and Radiological Sci. and Tech. (QST), 4 Health Research Institute, AIST

1

P-47

Ex vivo fluorescent imaging of macromolecule kinetics by nose-to-brain delivery withfunctional arginine-rich peptides
Takumi Kurano1,2 ,Takanori Kanazawa1,2, Mami Kaneko2, Hisako Ibaraki2, Yuuki Takashima2,
Toyofumi Suzuki1, Yasuo Seta2
1
School of Pharmacy, Nihon University, 2Tokyo University of Pharmacy and Life Sciences

─ 66 ─P-1
Whole-organ cell profiling accelerated by CUBIC combined with MOVIE imaging
Tomoki T. Mitani1,2, Katsuhiko Matsumoto1, Shuhei A. Horiguchi1, Junichi Kaneshiro1, Tatsuya C.
Murakami3, Tomoyuki. Mano3, Hiroshi Fujishima1, Ayumu Konno4, Tomonobu M. Watanabe1,
Hirokazu Hirai4, Hiroki R. Ueda1,3*
1
RIKEN Ctr. For Biosystems Dynamics Res., 2 Osaka Univ. Hosp., 3Grad. Med., Univ. Tokyo, 4 Grad. Med.,
Gunma Univ.

OBJECTIVE
Whole-organ cell profiling pipeline combined with CUBIC tissue clearing technologies is an effective tool to obtaincomprehensive cell information throughout the organ. Recently, we reported a single-cell-resolution whole-brain atlas(CUBIC-Atlas), which was novel scalable and sharable format for handling the whole-organ data by a point cloud ofcells. However, the positions of all detected cells in an organ had to be extracted from a massive image data to compact
the data size, and therefore the throughput of data acquisition and analysis remains a critical bottleneck for biomedical
applications handling numerous samples.
METHODSWe developed a high-speed volumetric imaging system named as MOVIE (MOVIng observation with Efficientreal-time autofocus) system for efficient volumetric imaging, which has integrated 1) continuous acquiring(MOVIE-scan), 2) real-time adaptive autofocusing (MOVIE-focus) and 3) skipping of blank images (MOVIE-skip).
MOVIE system was integrated into a custom-built light-sheet fluorescence microscopy. To analyze the large-scale
imaging data in a rapid timescale, we also developed a cell detection algorithm based on three-dimensional
Hessian-based Difference of Gaussian and a highly parallelization of multiple CPUs and GPUs.
RESULTS
The acquisition time was shortened to within a few hours per mouse whole organ. For example, using a ten-fold
expanded adult mouse whole-brain sample, the throughput of 50 ms of exposure time was measured as 0.56 TB/h andtotal data size was 6.3 TB using 10x objective lens with novel imaging system, comparing with 0.15 TB/h and 14 TB
with previous imaging scheme (Murakami et al., 2018). Acquisition time per brain also became 8.5 times shorter thanthe previous report. Our cell-detection algorithm worked at 1 hour/TB and had over 90% F-scores of nuclear stained cell
detection throughout the brain. Using a brain transduced with AAV-PHP.eB (NSE-H2B-mCherry) counterstained by
nuclear stain dye, we demonstrated the usability of the developed imaging and analysis pipeline by quantification ofmCherry positive cell number in each anatomical region and the ratio of positive cells in each 80 μm voxel. These results
showed that the cortex and hippocampus showed higher positive ratio than other areas.
CONCLUSIONS
Thus, the pipeline realizes a novel platform for the high-speed organ-scale analysis of cells and will promote
next-generation biomedical research targeting a large number of samples in a practical timescale. This content will be
published in Nature Protocols.

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P-2
Real-time cellular imaging of lung T-lymphocyte accumulation and focus formation ina mouse asthma modelAkihiro Hasegawa,1* Hidetaka Ogino,1 Toshinori Nakayama2
1
Department of Microbiology and Immunology, Yamaguchi University Graduate School of Medicine,2
Department of Immunology, Graduate School of Medicine, Chiba University
OBJECTIVE
The cardinal features of acute asthma are airway inflammation predominated by eosinophils, hypersecretion of mucus,
and airway hyperresponsiveness. A critical role for CD4+ Th2 cells in the pathogenesis of acute asthma has been
demonstrated in the studies of human asthma as well as of animal models of asthma. Th2 cell migration into the lung is
crucial for the initiation of asthma phenotype, but the in vivo dynamics of this process in living animals are poorly
understood since it has been difficult to visualize this process. Our aim was to image the cellular dynamics of the
migration of Th2 cells into the lung of living animals in a mouse model of asthma and identify the cellular processes
required for the initiation of the asthma phenotype.
METHODSWe developed a new in vivo color-coded real-time cellular imaging model using green fluorescent protein (GFP) andred fluorescent protein (RFP) transgenic CD4 T cells to visualize the dynamics of migration of T cells in a mouse modelof asthma.
RESULTS
Selective accumulation of antigen-specific CD4 T cells in the lungs was quantitatively imaged. The inhibition of
accumulation by dexamethasone was imaged. Accumulating GFP+ Th2 cells formed foci in the lungs from 6 to 20 hours
after antigen inhalation. The migration of CD69-deficient CD4 T cells into the asthmatic lung was severelycompromised. This process was also inhibited by the administration of anti-ICAM-1 or anti-VCAM-1 mAbs. Two days
after inhalation of antigen, GFP+ Th2 cells were detected in the area of eosinophil infiltration.
CONCLUSIONS
Focus formation generated by accumulating antigen-specific Th2 cells in the lung appeared to be a critical process in
the initiation of asthma phenotype. This new model enables the study of in vivo cell biology of airway inflammation andnovel drug discovery for lung inflammatory diseases.

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P-3
From Nano to Macro:
Advanced Imaging Techniques for Neuroscience Applications
Shinya Komoto*, Paolo Barzaghi, Koji Koizumi, Toshiaki Mochizuki, Bruno Humbel(OIST Imaging Section)
Imaging Section, Research Support Division
Okinawa Institute of Science and Technology (OIST)
1919-1 Tancha, Onna-son, Kunigami-gun
Okinawa, Japan 904-0495
OBJECTIVE:
An integrated approach with an array of different sample preparations, imaging technologies andsoftware analysis has been applied to tackle difficult tasks in Neuroscience.
The combination of classical laser scanning fluorescence microscopy with light-sheet microscopy and superresolution imaging techniques has been used to better understand the cellular mechanisms and neural circuitry
underlying learning and adaptive behavior in the brain.
METHODS & RESULTS:
Cholinergic system plays a fundamental role in mammal’s brain and is involved in neurodegenerative
diseases, thus a characterization of cholinergic neurons (number, size and spatial distribution) might contributeto a better understanding of neuronal disorder like Alzheimer. The use of fast laser scanning confocal coupled
to high sensitive and with a better spatial resolution detector (Airyscan detector, Zeiss Microscopy) allowed
the collection of large volumetric data fulfilling the Nyquist criteria but still maintaining a single cell resolution
in an acceptable time frame.
To further develop our imaging techniques to understand the cellular mechanisms of neuronaldevelopment, we combined the use of clearing protocols and high-speed confocal system (e.g. light sheet
microscopy, multi-point laser confocal system). The latter allows the imaging of large volume specimen. In
particular, we have focused on visualizing microglia, the professional phagocytes of the brain serving bothimmune and glial functions. In details, we have image the brain together with the microglia and determine thenumber and distribution of microglial cells in post-injured brain.
CONCLUSIONS:In this work, it has been shown that the complexity of cellular mechanisms and neural circuitry of
the brain can be better understand using not only a single approach but an ensemble of different techniques
and technologies.

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P-4
New evaluation technique for micro-environment
using translational and rotational diffusion
Johtaro Yamamoto,1,* Akito Matsui,2 Masataka Kinjo 2
Biomed. Res. Inst., AIST, 2 Fac. Adv. Life Sci.., Hokkaido Univ.

1

OBJECTIVE
Macromolecular crowding (MMC) in cells is recently an attractive issue in biology field. However, there are no
promising measurement method for the degree of crowding yet. We developed a polarization-dependent fluorescencecorrelation spectroscopy (Pol-FCS), which enables to measure rotational diffusion and translational diffusion offluorescent molecules simultaneously. We found the Pol-FCS is capable of evaluation for extent of space, namely
microenvironment surrounding target fluorescent molecule. In this work, we suggest a new evaluation technique formicro-environment such as MMC using Pol-FCS.
METHODS
Figure 1 shows a sematic diagram of Pol-FCS system. An
excitation laser with a wavelength of 488 nm is reflected bya dichroic mirror and focused into a sample droplet by anobjective lens. The droplet is an MMC solution or othersolution of 100 nM EGFP. The fluorescence of EGFP in thedroplet pass through the dichroic mirror and emission filter.
The fluorescence separated into 4 beams by a polarizing
beam splitter and two non-polarizing beam splitters. Eachfluorescence beam was coupled to multimode optical fibers
with a core diameter of 62.5 m acting as pinhole, andfinally detected by four avalanche photo diodes.
Cross-correlation functions of recorded fluorescenceintensity signals were obtained using hardware correlator.
The characteristic decay time of the cross-correlation
functions were analyzed by non-linear least squares method
using theoretical cross-correlation function of Pol-FCS.

SampledropletCover slip

Objective

Laser
Fluorescence
Emission filter
NPBS

MMF1

PBS
L1

L2
NPBS

MMF4
L4

L3

MMF2

MMF3

Fig. 1. Experimental setup. L: Lens. MMF:
multi-mode optical fiber. PBS: polarizing beam splitter,
NPBS: non-polarizing beam splitter.

RESULTS
We compared the characteristic decay time of rotational and translational diffusion (rotational diffusion time andtranslational diffusion time) of EGFP in various MMC solutions and other solutions. In sucrose solution of phosphatebuffer saline (PBS), which is small molecule solution, the ratio of rotational diffusion time and translational diffusion
time was always 1 independent of the concentration of sucrose. This was reasonable result because both the rotational
and translational diffusion coefficients are inversely proportional to the viscosity of solution according to the
Stokes-Einstein relationship and the Debye-Stokes-Einstein relationship. On the other hand, in the MMC solutions suchas ficoll or bovine serum albumin in PBS, the ratio was lower than 1, suggesting that the rotation of EGFP molecule iseasier than the translation in MMC solution. This indicates the translational diffusion can be disturbed by surrounding
crowder molecules, however the EGFP molecules can rotate within gap between crowder molecules.
CONCLUSIONSIn this work, Pol-FCS measurements in MMC solutions were performed and the rotational and translational diffusion
were compared. The results indicating that such the comparison gives information about micro-environment aroundtarget molecules including MMC condition. We expect Pol-FCS will be a useful tool to quantitatively evaluate MMC.

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P-5
Simultaneous Tracking of Multiple Biomolecules in a Living Filamentous Fungus by
Confocal Raman Microspectroscopic Imaging
Mitsuru Yasuda,1 Norio Takeshita,2 Shinsuke Shigeto 1,*
School of Science and Technology, Kwansei Gakuin University, Japan
2
Faculty of Life and Environmental Sciences, University of Tsukuba, Japan
1

INTRODUCTION: A filamentous fungus known as a mold is composed of thread-like long hyphae, and grows by
elongating the tips of the hyphae. In order to investigate the elongation mechanism of the hyphae, we focus on confocal
Raman microspectroscopic imaging, which gives spectral information reflecting the molecular structure of biomolecules
in a cell in vivo and the information on their spatial distributions [1]. Additionally, an integrated approach with stable
isotope labeling can be useful for further investigation of the elongation mechanism. The Raman band of a substance ofinterest with a heavy stable isotope shifts to lower frequency because of the isotope effect. By monitoring this shiftedband, the behaviors of multiple intracellular biomolecules associated with metabolism of stable isotope-labeled
substances can be tracked simultaneously [2]. Therefore, this integrated approach is expected to provide new insight into
the elongation mechanism of the fungal hypha from the viewpoint of metabolism. In this presentation, we report asimultaneous tracking of multiple biomolecules in a living filamentous fungus by stable isotope-labeled confocal Raman
microspectroscopic imaging.
MATERIALS AND METHODS: Aspergillus nidulans was used in this work, which is widely used as a model
filamentous fungus. As stable isotope-labeled substrates, D (deuterium)-labeled glucose and 13C-labeled glucose wereused. A. nidulans was first cultured in a liquid medium containing the unlabeled glucose instead of D-/13C-labeled
glucose at 25 °C for 1–2 days. After removing the medium, a fresh liquid medium containing D-/13C-labeled glucose
was then added. Raman imaging experiments of hyphae were performed with a confocal Raman microspectroscopic
system (XploRA Nano, HORIBA) while cultivating A. nidulans.
RESULTS AND DISCUSSION: Since the Raman spectrum of a cell generally contains Raman bands of various
molecules, it is significantly difficult to decode what kind of components Raman imaging data consists of. To firstinvestigate main components constituting Raman imaging data, multivariate curve resolution-alternating least-squares(MCR-ALS) was employed [1]. MCR-ALS allowed us to reveal that the Raman imaging data of A. nidulans hyphae aremainly composed of proteins including cytochromes, nucleic acids, polysaccharides, and lipids. The spatial distributionsof these intracellular biomolecules were visualized simultaneously [3]. Based on these results, experiments using stable
isotope-labeled substrates were then performed. When A. nidulans were grown with D-labeled glucose, molecules
whose hydrogen atoms were substituted with D were localized along the edge of the hyphal tip. This localizationsuggests that D-labeled glucose was taken up selectively from the apex of the hypha. Moreover, experiments using
13
C-labeled glucose enabled us to track the biosynthetic process of reduced cytochrome b/c, and showed that metabolic
activity is higher at the apex of the hyphae and the inner part of hyphal tips than a part away from the apex and the outerpart of hyphal tips. The elongation mechanism inferred from the obtained results is as follows: (i) Glucose is taken up
selectively from the apex of the hypha by glucose transporters. (ii) The incorporated glucose diffuses along the edge ofthe hyphal tip. (iii) Metabolic activity around the tip increases as a result of the high glucose concentration. (iv)
Substances necessary for the elongation are synthesized at around the hyphal tip consuming the glucose. (v) The hypha
elongates. In conclusion, we have proposed a new hyphal elongation model based on glucose metabolism in this work.
ACKNOWLEDGEMENTS: This work was supported by JST ERATO Grant Number JPMJER1502 (Japan) and the
Research Foundation for Opto-Science and Technology (Japan).
REFERENCES:[1] C.-K. Huang et al., Anal. Chem. 84, 5661 (2012).
[3] M. Yasuda et al., Anal. Chem. submitted.

[2] H. N. N. Venkata et al., Chem. Biol. 19, 1373 (2012).

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P-6
Attempt to observe the structure of schizophyllan monomer by Small Angle X-ray
Scattering and Molecular Dynamics simulation
Yoshitaka Matsumura and Masaki KojimaSch. of Life Sci., Tokyo Univ. of Pharm. and Life Sci.
OBJECTIVE
β-glucan is a major component of the pathogenic fungal cell walls. Depending on their composition and structure, it isrelated to diseases like mycoses and shows good effects as health promotion such as anti-tumor effect etc. Since
β-glucan is a target molecule for candin antifungal drugs, it is an important molecule from the viewpoint ofstructure-activity relationship of antibacterial drugs. Dectin-1 and the like have been found as receptors for β-glucan, andare positioned as important molecules also from the viewpoint of infectious immunity and innate immunity.
Schizophyllan, which is a β-glucan derived from Schizophyllum commune, has been approved as an anticancer agent
because it exhibits antitumor activity, and it has been clarified by X-ray crystal structure analysis that it dissolves in water
to form a triple helix structure (Takahashi, Y., et al. 1984, Kony, D. B., et al., 2007). In contrast, it is known thatsonication of schizophyllan reduces the molecular weight and causes a structural change from trimer to monomer,
resulting in loss of anticancer activity (Yanaki, T., et al. 1983). However, it has been found that the monomericschizophyllan exhibits activity in the horseshoe crab fluid coagulation reaction (Limulus test, Nagi, N., et al. 1993). The
Limulus test has been used since 1980's to determine mycosis, a major human disease. However, there is no example inwhich the structure directly was observed in the solution.
Previously, we have used Physicochemical methods such as Small Angle X-ray Scattering (SAXS), Atomic ForceMicroscopy (AFM), solution Nuclear Magnetic Resonance (NMR), and molecular dynamics (MD) simulation toinvestigate the three-dimensional structure of β-glucan derived from Agaricus brasiliensis. The results indicated that the
β-glucan derived from Agaricus brasiliensis in aqueous solution is multi-dispersed state, trimers and/or monomers, theshape of the molecule is globule, the main axis of β-1,6 bond and the side chain of β-1,3 bond (10%) (Matsumura, Y. &Kojima, M., 2018). So, in the present study, we attempted to observe and visualize the three-dimensional structure of themonomeric schizophyllan molecule treated with sonication by SAXS and MD simulation.
METHODSThe SAXS experiments were carried out on beamline BL-6A at the Photon Factory. All data were collected using
X-ray of wavelength of 1.5 Å with a PILATUS3 1M dectector (Dectris). The sample-to detector distance was 0.5 m.
Equilibrium experiments were performed at 25℃. The SAXS intensities were accumulated for a total of 3.0 s byrepeating the measurements for a period of 0.1 s each time in order to ensure enough statistical precision. X-ray
scattering data were obtained from schizophyllan and the corresponding aqueous solution. The scattering data of theaqueous solution were subtracted from those of the schizophyllan solutions. X-ray scattering data were analyzed by
Guinier approximation, as assuming an exponential dependence of the scattering intensity on h2, where h = 4πsinθ/λ andθ is half of the scattering angle (Guinier, A. and Fournet, G., in Small Angle Scattering of X-rays. Wiley New York,1995. The radius of gyration (Rg) and zero angle scattering intensity I(0) were determined using Guinier approximation(Guinier, A. and Fournet, G., Small angle scattering of X-rays. Wiley, 1995).
Molecular dynamics simulation was permormed by AMBER. The details of the simulation are written in the poster.
RESULTS
By SAXS analyses showed that schizophyllan treated with sonication was multi-dispersed state, and the shape was
globule. The Rg of major component of the schizophyllan at several concentrations were almost 10 Å. MD simulation of
the schizophyllan suggested that the shape was helical structure. As a results, it is suggested that the possibility which theschizophyllan is a good model showing biological activity in any structural form of the trimeric helical structure,
monomeric helical structure, and random coil. The details are discussed in the poster.
─ 72 ─P-7
Structural basis for the substrate specificity of phosphoenolpyruvate carboxykinases asphosphodiester energy-conversion enzymes
Takuya Miyakawa,1,* Yoko Chiba,2 Yasuhiro Shimane,2 Masaru Tanokura1
1
Grad. Sch. Agric. Life Sci., Univ. Tokyo, 2D-SUGAR, JAMSTECOBJECTIVE
Inorganic pyrophosphate (PPi) is the simplest compound containing a high-energy phosphate bond between two
phosphate (Pi) molecules. PPi can act as an energy and phosphate donor similar to nucleoside di- or tri-phosphates,
including adenosine triphosphate (ATP), in cellular reactions. Several enzymes selectively utilize PPi over ATP and othernucleotides to catalyze similar reactions as ATP-dependent functional homologs. Because PPi has the simplest structure
among molecules with a high-energy phosphate bond, this molecule has been proposed as an evolutionary precursor ofATP. Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of thecentral metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. ATP- and
GTP-type PEPCKs were well established to their enzymatic properties including the selectivity of phosphate donor.
More recently, PPi-type PEPCK was characterized, which clarified that the amino acid sequence similarity of these
PEPCKs was too low to confirm that they share a common ancestor [1]. Here, we attempted to elucidate how the
PPi-type PEPCK gained the selectivity of PPi as a phosphate donor and what evolutionary relationship the enzyme haswith ATP- and GTP-types.
METHODS
PPi-type PEPCK form Actinomyces israelii (AiPEPCK) was produced using the Escherichia coli expression system
and was then purified by a series of column chromatography experiments. X-ray diffraction data were collected from thecrystals of the purified enzyme on the beamline BL-17A at Photon Factory (Tsukuba, Japan). The crystal structure of
AiPEPCK was determined at 2.6 Å resolution by using a single-wavelength anomalous dispersion (SAD) method.
RESULTSAiPEPCK adopted a core structure enveloped by some lobe structures (Lobes 1 to 4). The core structure was divided
into two globular α/β domains, N-terminal domain and C-terminal domain. The Co2+ ion located in the deep cleft
between two domains, indicating that the cleft probably functions as active site of AiPEPCK. The structural
superposition with ATP- and GTP-type PEPCKs revealed that AiPEPCK shared a core structure and all of the catalytic
residues which directly interact with Co2+ and Mg2+ ions, γ-phosphate group of ATP or GTP, PEP and oxaloacetate inATP- and GTP-type PEPCKs. On the other hand, the residues in the purine-binding region were not conserved among
three types. Especially, the bulky side chain of His920 sterically hinders the access of ATP or GTP to the active site in
AiPEPCK. In addition, the core structure of AiPEPCK was fixed to a closed conformation due to the dimer formation bycontacting the AiPEPCK-specific appendage structures, Lobes 2 and 3, which contribute to narrow the purine-binding
regions for the PPi selectivity.
CONCLUSIONSThe PPi selectivity of AiPEPCK requires the presence of His920 with bulky side chain in purine-binding region anddimer formation between Lobes 2 and 3. Furthermore, the order of secondary structure elements and residues directly
involved in enzymatic function were conserved between these PEPCKs, indicating that they share a common ancestor.

[1] Chiba et al., Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria. J. Biol.
Chem., 290, 23960‒23970 (2015)

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P-8
Analysis of myosin II localization during cytokinesis of sea urchin eggs
using super-resolution microscopy
Natsumi Hosoya1,*, Issei Mabuchi2
1
Faculty of Social Information Studies, Otsuma Women’s University,2
Department of Life Sciences, Graduate School of Arts and Sciences, The University of TokyoOBJECTIVE
During cytokinesis of animal and fungal cells, the actomyosin contractile ring (CR) is formed at the division site anddivides the cell into two by its contraction. Non-muscle myosin II is known to be concentrated in the CR and essential
for both formation and contraction of the ring, but the precise localization of myosin II in the CR remains unsolved.
We have been studying structure and function of the CR during cytokinesis of echinoderm eggs, since these eggs
divide synchronously and the cell cortex including the CR (cleavage furrow) can be isolated.
Here, we investigated myosin II localization using antibodies raised against sea urchin egg myosin using confocal
microscopy and super-resolution microscopy.
METHODS
Cortices containing CR were isolated from dividing sea urchin (Hemicentrotus pulcherrimus) eggs as previously
described (Mabuchi 1994). Actin filaments were stained with Rhodamine-phalloidin. Indirect immunofluorescencemicroscopy was performed according to Mabuchi (1994) using rabbit polyclonal antibodies raised against myosin heavy
chain (HC) of sea urchin (Tripneustes gratilla) egg and mouse monoclonal antibodies raised againstmonophosphorylated myosin regulatory light chain (1P-MRLC). Confocal microscopy was carried out using
Olympus FV1000 laser scanning microscope. Super-resolution images were obtained by N-SIM (Nikon Structured
Illumination Microscopy).
RESULTS and DISCUSSION
Both actin and myosin II were localized to the division site of isolated cortices from the very beginning of formation
till the full contraction of the CR as seen by confocal microscopy. The actin staining was observed just beneath theplasma membrane (PM) to about 2.0 µm toward cytoplasm. Myosin II-positive layer was seen to be thinner than theactin staining and found at a little deeper region, 1.0 to 2.0 µm away from the PM, than the actin staining.
Next, the staining pattern of myosin II was examined using either anti-myosin HC or anti-1P-MRLC by N-SIM.
Myosin HC staining was strong at the cytoplasmic side of the CR. Furthermore, the staining of 1P-MRLC was foundin the innermost region of the CR. This specific localization of myosin, especially its activated form, in the CR has
never been reported previously. This may suggest that myosin II may interact with actin filaments to drive CR
contraction at a region a little distant from the PM, but not just beneath the PM.
It has been shown by electron microscopy that purified sea urchin egg myosin II forms bipolar filaments in vitro(Mabuchi, 1973). The length of the filaments was reported to be 370±20 nm. We found that myosin II (myosin HC)
in the CR observed by N-SIM formed a structure similar to the bipolar filament. We picked up such images andmeasured their length using the imaging software (Image J). The average length of 20 images was 394±40 nm, which
agreed well with that of the bipolar egg myosin II filaments formed in vitro. This result suggests that bipolar myosin
filaments may play a key role in formation and contraction of the CR in dividing sea urchin eggs.
REFERENCES
Mabuchi, I. (1973) J. Cell Biol. 59, 542-547.
Mabuchi, I. (1994) J. Cell Sci. 107, 1853–1862.

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P-9
Measurement resolution of diffraction phase microscopy
as investigated with reflection interference microscopy
1

Shota Ueki,1 Hisashi Hujiwara 1,*
Graduate School of Information Sciences, Hiroshima City University

OBJECTIVE
Diffraction phase microscopy (DPM) employs a common-path interferometer robust against the mechanical vibrationand provides quantitative phase images related to both thickness and refractive index of transparent samples such asliving cells [1].
To ensure the phase resolution of DPM, transparent standard samples with well-defined thickness steps have beenfrequently used; the step sizes are usually larger than 100 nm [2]. Instead of such samples, we propose to use a soap film,
because soap films are known to exhibit stepwise thinning called “stratification” where each step is about 10-20 nm [3].
The thickness of a soap film can be monitored with reflection interference microscopy (RIM). Therefore, simultaneous
application of DPM and RIM to the soap film during stratification enables us to investigate precise phase resolution of
DPM.
METHODS
We have constructed a simultaneous measurement system of both DPM and RIM. The system includes an objective(5 or 20) and dichroic mirrors. The epi-illumination light source (405 nm) for RIM is a combination of a mercury
lamp and an interference filter. For DPM, on the other hand, the transmission light source (682 nm) is asuperluminescent diode (FOLS-04, Craft Center SAWAKI). A quantitative phase imaging unit (QPI-U1, Pi Photonics) isused for making interference fringe images, from which the phase delay as a result of the light transmission through thesample can be extracted by two dimensional Fourier transform analysis [4].
To obtain simultaneous images of both DPM and RIM, we have constructed an image recording system where two
CCD cameras (SXC10, Baumer) synchronously acquire the images at 100 fps with the synchronization signal from asignal generator.
RESULTSWe recorded the simultaneous DPM and RIM images of a vertical soap film made from 0.1 M sodium oleate aqueous
solution. Detailed analysis of both simultaneous images is now still in progress, but even brief analysis of the RIM
images confirms that the stratification actually occurred in the observed soap film, which supports the use of a thinningsoap film to investigate the phase resolution of DPM.
CONCLUSIONS
We have constructed a system to acquire and record simultaneous DPM and RIM images. The detailed analysis ofboth DPM and RIM images of a soap film during stratification is expected to reveal the phase resolution of DPM.
REFERENCES[1] G. Popescu et al., Opt. Lett., 31, 775 (2006).
[2] T. H. Nguyen et al., Opt. Lett., 39, 5511 (2014).
[3] A. A. Sonin and D. Langevin, Europhys. Lett., 22, 271 (1993).
[4] M. Takeda et al., J. Opt. Soc. Am., 72, 156 (1982)

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P-10
Development of evaluation method of cell morphology using Digital In-line
Holographic Microscope (D-IHM)
Shuhei Yamamoto,1 Ryuji Sawada,1 Toru Ezure,1 Kanna Nagaishi 2
Analytical & Measurements Division, Shimadzu Corporation, 2Second Department of Anatomy, Sapporo
Medical University.

1

OBJECTIVE (11pt)
In regenerative medicine and cell therapy, the importance of non-invasive evaluation of large numbers of cells isincreasing day by day. The most popular non-invasive method for morphological evaluation is the analysis of phase
contrast microscopy images, but it is impossible to quantitatively evaluate the three-dimensional shape of cells from phase
contrast microscopy images. Because the three-dimensional shape of the cell changes with the accumulation of
intracellular organelles and functional proteins, it is very important to evaluate the three-dimensional shape. We measured
the optical thickness, which is the product of the refractive index and the three-dimensional shape of the cell, using DIHM, which has a wide field of view and high resolution. We have developed a method to quantitatively evaluate thegradient of the optical thickness of the cell contour by focusing on the gradient of the cell contour as a shape indicating thestate of the cell.
METHODS (11pt)
Cells are irradiated with coherent light of different wavelengths. CMOS image sensor captures an interference patterncaused by the light passing through the cell and the reference light passing around the cell. Phase information of light isretrieved numerically by using 4 different wavelength images, and reconstructed image at cell surface is calculated by
wavefront propagation. The phase delay of thins transparent object, such as cells, are visualized by reconstructed phase
images. The brightness of the pixels of the phase image represents the phase change. So, the luminance value of each pixelof a differential image of a phase image indicates the magnitude of the inclination of optical thickness in each pixel.
Therefore, the histogram of the differential image shows the distribution of the gradient of the optical thickness, and it ispossible to quantitatively evaluate of which gradient of the optical thickness of the cells is steeper in the image.
RESULTS (11pt)
We evaluated the cell morphology of mesenchymal stem cells with different passage numbers. Cells with different
passage numbers had different three-dimensional cell morphologies, and our method was able to quantitatively evaluate
the differences.
CONCLUSIONS (11pt)
We developed a non-invasive and quantitative method to evaluate the optical thickness of a large number of cells using
D-IHM. This method is considered to be very useful as a technique for evaluating the cell state in the field of regenerative
medicine and cell therapy.

─ 76 ─P-11
Development of the imaging methods for phenotyping of knockout micewith the X-ray Computed Tomography
1

Hirotoshi Shibuya, 2 Taku Gotoh, 2 Ruriko Tanabe, 2 Shintaro Nomura, 1,* Masaru Tamura
1
RIKEN BRC, 2 Nagahama Inst. of Bio-Sci. and Tech.

OBJECTIVE
The genome editing technologies such as CRISPR/Cas9 system make it possible to the gene knockout of various
animals with unprecedented simplicity and speed. It means that bottleneck of the functional gene analysis will change
from generating the knockout animal to the phenotyping. Therefore, we need easy and high-throughput phenotyping
method. To overcome this issue, we have developed imaging techniques that used the X-ray computed tomography(CT) and contrast agents.
METHODS·Ex vivo X-ray CT imaging of the soft tissues
The tissue samples were fixed with suitable formalin-based fixatives and immersed in the contrast agents. After thecontrast enhancement, samples were mounted in the plastic tube and scanned with the high-resolution X-ray CT.
·Three-dimensional angiography using the new angiographic agent
Mice were perfused transcardially with contrast agent using a peristaltic pump under deep anesthesia. After theperfusion, tissue samples were isolated and scanned with the high-resolution X-ray CT.
RESULTSWe succeeded the development of imaging techniques to visualize the mouse soft tissues by X-ray CT at highthroughput and high-resolution (Fig.1). Moreover, we developed new angiographic agent, and detected whole vascularand capillary network without structural deformation (Fig.2).
CONCLUSIONS
These methods provide the quantifiable datasets to characterize pathological conditions in disease mouse models.
Simple X-ray CT
contrast
E14.5
embryoContrast-enhanced X-ray CT
Adult kidney
E14.5 embryo

Angiography

Glomerulus
Fig.1, CT images of the mouse soft tissues

─ 77 ─

Fig.2, Adult Kidney

P-12Open Source Local Gene Induction System by IR Laser Irradiation
Joe Sakamoto,1, * Yuko Kamikawa,1 Yasuhiro Kamei 1, 2
1 Spectrography and Bioimaging Facility, NIBB Core Research Facilities, NIBB,
2 School of Life Science, SOKENDAIOBJECTIVE
InfraRed Laser Evoked Gene Operator (IR-LEGO) is a spatiotemporal gene induction system via aheat shock response (HSR). HSR is well known as heat protective response of living organisms, and isconserved among various organisms. In HSR, heat shock transcription factor 1 is activated by heat treatment,
and promotes expression of a group of chaperone proteins, so called heat shock proteins. Because HSP
promoter is easily activated by heat stress, HSP promoter is traditionally used for temporal gene induction.
Although temporal gene induction is realized by HSP promoter, spatial control of gene expression is still
difficult. To overcome this problem, IR-LEGO utilizes not only HSR, a biological technique, but also
microscope, an optical technique. Since living organism is consisted from water, 1,480 nm IR laser which isabsorbed by water molecules is used to heat cells for activation of HSP promoter. And IR laser is focusedthrough objective to regulate spatial activation of HSP promoter. Thus, IR-LEGO realized spatiotemporal
induction of gene expression. To utilize IR-LEGO effectively, researchers can label single cells byfluorescent protein, and trace cell lineage. Also, researchers can express a functional protein ectopically.
Therefore, gain of function experiment can be performed with single-cell resolution. Furthermore, HSR isconservative system, IR-LEGO can be applied to various organisms such as medaka, zebrafish, Iberianribbed newt, A. thaliana, and so on. For these reasons, IR-LEGO has a possibilities to be powerful techniquefor various research fields. However, almost all research reports which use IR-LEGO is published fromJapanese researcher. Recently, a few foreign researchers published reports which use simple self-constructed
IR-LEGO system, but still limited. For those who is not professional of optics and microscopy, it is difficult
to construct IR-LEGO optical system. In the present study, we show how to construct IR-LEGO by one’s
self, and introduce its application.
METHODS
We constructed whole optical system of IR-LEGO including inverted microscope. Open source (os)IR-LEGO consists of two optical component, inverted microscope and IR-LEGO optics. In IR-LEGO optics,
IR laser was collimated and expanded its beam diameter, and was introduced to inverted microscope
component. In inverted microscope optics, IR laser was introduced to objective by a dichroic mirror, andemitted to samples through objective. To adjust an IR laser focal point, we used specific ink pen (Kawakimapen from Shachihata co., Ltd.) or up-conversion particle. To confirm that osIR-LEGO works heat shockreporter transgenic medaka and cell line was used. Transgenic medaka is expressed Venus fluorescent protein
which driven from hsp promoter. Heat shock reporter cell line stably has Venus or EGFP driven by hsp
promoter. To use these transgenic medaka and cell line, we could visualize whether HSR was activated ornot.
RESULTS
Because of chromatic aberration of objective, focal point of IR laser is different from visible light.
We adjust the focal point of IR laser visualized by specific ink pen or up-conversion particles. We
successfully constructed osIR-LEGO. We could also observe fluorescent protein expression as a reporter ofheat shock response in both in vivo and in vitro by irradiating target region using osIR-LEGO.
CONCLUSIONS
Although IR-LEGO has potential abilities to apply to various fields of research, publications which
effectively utilizes IR-LEGO are still limited. One problem is that it is difficult for foreign researchers,
especially who are not professional of optics, to obtain IR-LEGO. The other is that there are no protocols
which describe how to construct IR-LEGO precisely. In the present study, we demonstrated that IR-LEGO
can be constructed by one’s self. This research will be useful for the potential users of IR-LEGO.

─ 78 ─

P-13
Microwave-antenna-integrated cell dishes for nanodiamond quantum thermometry
Keisuke Oshimi1, Yushi Nishimura1, Masuaki Tanaka2,
Eiji Shikoh 2, Masazumi Fujiwara1, Yoshio Teki1
Grad. Sch. Sci. of Osaka City Univ.1, Grad. Sch. Eng. of Osaka City Univ.2
OBJECTIVE
Fluorescent quantum nanodiamond thermometry is a promising new technology that may enable nanoscale temperature
measurement inside cells with a high sensitivity down to milli-Kelvin. In this technique, microwave irradiation to cell isunder the optical microscope to exploit optically detected magnetic resonance (ODMR) of luminescent color centers,
namely nitrogen vacancy (NV) centers. In order to carry out this operation simply and quickly, we have recently developed
culture dishes equipped with microwave antenna [1], where the linear antenna structure was used. However, the linear
antenna allowed us to measure ODMR only in a narrow apace range, 100 μm distance from the antenna (Fig. 1(a)), which
is a significant drawback for the efficient measurement. In this study, we have developed a wide and broadband microwave
antenna that enables observation of ODMR in the wide area on cell culture dishes.
RESULTS
The antenna structure was designed based on a split-ring resonator concept having the resonance frequency of near 2.87
GHz required for the ODMR. We used a finite element method simulation for the antenna design. Figure 2 shows a microscope photograph of cultured-cells with the linear microwave antenna on our dishes. Figure 1(b) shows simulated intensity distribution of the magnetic and electric fields of this antenna. The irradiated area was expanded 10 times or morethan that of the conventional linear structure. We also found that it was possible to control the spatial distribution of theelectric field by this resonator, which may be toxic to cells. When the conventional linear structure was used, the magneticfiled spatial distribution was overlapped with the electric field, so that cells were subjected to the electric field irradiation.
In the new structure, they do not overlap each other and cells may avoid the electric field irradiation. Figures 2 and 3 showa photograph of cultured cells near antenna structures fabricated by photolithography the simulated spectrum of the reflection coefficient of the antenna. It has a resonance peak at around 2.75 GHz, which means the microwave can be irradiatedmost efficiently at this resonance point.
CONCLUSIONSThe split ring resonator microwave antenna was designed on culture dishes for the large-area microwave irradiation. The
structure was fabricated by photolithography and used for cell culturing.
(a)

S11 [dB]

0

(b)

-2
-4
-6
-8

-10
2.4

Figure 1. Comparison of linear
antenna (a) and antenna with split
ring resonators (b).

Figure 2. A microscope photograph of cultured-cells near theantenna.

2.6
2.8
3.0
Frequency [GHz]3.2

Figure 3. Calculation of reflection coefficient of the antenna structures.

[1] H. Yukawa, M. Fujiwara, K. Kobayashi1, Y. Umehara, Y. Kumon, K. Miyaji, T. Iwasaki, M. Hatano, H. Hashimoto,Y. Baba, submitted.
─ 79 ─

P-14
Titanium Dioxide Nanoparticle is a Brilliant Perfusion Contrast Agent for
Visualization and Diagnosis of Small-Animal Microvessel.
1 Ruriko Tanabe, 1 Taku Gotoh, 2 Hirotoshi Shibuya, 2 Masaru Tamura, 1,*Shintaro Nomura
1

Nagahama Inst. of Bio-Sci. and Tech., 2 RIKEN BRC

OBJECTIVE
It is important to develop highly sensitive contrast agent for visualization and quantification of microvessels insmall-animals for preclinical research. Commonly available contrast agents have problems such as rapid clearance
and leaking out from vessels due to the small size of molecule, deform the vessels by high pressure in perfusion due totheir high viscosity, and stuck the small vessels by the aggregation particularly in small animals. Nanoparticle oftitanium dioxide with dispersing reagent, which would dissolve the problems, was examined as promising
contrast-enhancing agent in the study.
METHODS
Deeply anesthetized 8 weeks-old mice were perfused with saline (0.9% NaCl) until red-blood cells are not visible.
Then nanoparticle of titanium dioxide solution (generously gifted by TAYCA Corp.) containing dispersing reagent was
injected left ventricle to visualize systemic arteries of brain, kidney, digestive organs, liver and eye, or right ventricle tovisualize pulmonary artery.
Dissected organs were analyzed by micro-computed tomography (micro-CT).
Three-dimensional reconstitution of CT-imaging was performed by OsiriX software.
RESULTSAs shown in the pictures below, arteries of Brain (left) and Liver (right) were clearly visualized up to 10
micrometers diameter in size without deformation. Furthermore, bleeding area was detected in experimental model ofacute hepatitis, and disruptions of arteries were detected in the model of thrombosis.

CONCLUSIONSThe perfusion contrast agent developed in this study gave us sensitive and accurate results in angiogram and will
be useful for diagnostic research in preclinical field (1, 2).
1. Shibuya H. et al. The 6th International Symposium on Bioimaging. 2019.
2. Shibuya H. et al. Annual Meeting of Genetic Soc. Japan. 2019.

─ 80 ─

P-15
Multimodal OTN-NIR/MRI imaging with IR-1061@Gd-DOTA imaging molecules
Doan Thi Kim Dung1,4, Masakazu Umezawa2, Karina Nigoghossian2
Gil Yeroslavsky3, Kyohei Okubo2,3, Masao Kamimura2,3, Masayuki Yamaguchi4, Hirofumi Fujii4, andKohei Soga2,3
1
Research Institute of Biomedical Science, Tokyo University of Science, 2 Department of Material Scienceand Technology, Tokyo University of Science, 3 Imaging Frontier Center (IFC), Research Institute for Scienceand Technology (RIST), Tokyo University of Science, 4 Exploratory Oncology research & Clinical Trial center,
National Cancer Center Hospital East, Japan.
OBJECTIVE
In recent, multimodal imaging has attracted an enormous attention from researchers in various fields. The
combination of individual techniques is expected to provide a comprehensive image from the objects [1]. Among
medical imaging techniques currently exploited, magnetic resonance imaging (MRI) is considered the most beneficial
one because it is a non-ionizing radiation technique which provides images with high resolution, high tissue contrast as
well as great signal-to-noise (SNR) ratios. While optical imaging can obtain functional or molecular information, theoverlaying of optical images onto an anatomical map provided by MRI enables the overlaying of optical images on
to anatomical maps provided by MRI can promote comprehensive understanding of the object frommolecular to anatomical level. Optical imaging with the near-infrared over 1000 nm (OTN-NIR) emission under
980nm excitation is growing as a promising technique because the light has its maximum depth of penetration throughbiological tissue from several millimeters to centimeters thickness [2, 3] utilizes the so-called biological window [2]. In
this work, IR-1061, a molecule emitting over 1000 nm fluorescence under 980 nm excitation is employed as an imaging
probe. The probe is then trapped in a micelle composed of PEG-based co-polymer conjugated Gd-DOTA, an MRI
contrast agent.
METHODS
Preparation of IR1061@PEG-co-block-polymer@Gd-DOTA probe
170µL of Gd-DOTA solution 17 mg/mL was added in 1 mL of MES buffer at pH 5.5 and mixed and after 5 minutes,
300 µg of EDC and 500µg of NHS were alternately added into the mixture. Finally, 1.5 mg of PEG-co-polymer-NH2 in
1 mL of MES buffer, pH 5.5 and incubated for 6 hours. The conjugated polymer was dissolved in 1 mL of acetonnitrile(ACN) and 4 mL of water. 40µL of IR1061 in ACN (0.5 mg/mL) was then added to the mixture and stirred overnight.
RESULTSNIR absorption and emission and imaging
Absorption spectra of the probe was determined by an UV-VIS-NIR v770
spectrometer showed the absorption range from 800-1150 nm with the highest
peak around 1050 nm. Emission spectra were obtained by Avaspec-NIR 256-1.7
spectrometer showed the sharp emission around 1200 nm. Size distribution was
obtained by DLS and the size distribution is 22 nm. The probe was then injected
into mice and observed the NIR imaging by SAI-1000 system and NIR
microscope.
T1 contrast effect
T1 enhancement effect of the probe was confirmed on spin-echo T1-weighted
images (Fig. 2) and imaging with phantom was conducted with a 3 tesla
whole-body scanner (Signa HDx 3T; GE Healthcare). The molecular probe
Fig.1: Emission spectrum of the probe.
exhibited an enhancement on T1 relaxation as in phantom.
Further animal images by both OTN-NIR/MRI will be presented at theconference.
CONCLUSIONS
The multimodal imaging OTN-NIR/MRI is able to materialize with a favor of
IR1061@PEG-based-co-polymer@Gd-DOTA multimodal imaging probe.
Further functionalization with antibodies heralds advances in areas of cancer Fig.2: T1-weighted images of phantoms
containing various concentrations ofdiagnosis.
IR-1061-Gd-DOTA probe (TR = 200
References
ms; TE = 9 ms).
[1] K Shah, Gene Therapy 11, (2004) 1175–1187.
[2] A Schmidt, Nat Nanotechnol. 4 (11), (2009) 710-1.
[3] E Hemmer, Nanoscale Horiz. 1, (2016) 168.
─ 81 ─

P-16
Development of Functional 19F MRI Contrast Agents
Using Perfluorocarbon-Encapsulated Nanoparticle
Yuki Konishi1, Kazuki Akazawa1, Rena Akimoto1, Fuminori Sugihara2,3,
Masafumi Minoshima1, and Kazuya Kiuchi1,21Graduate School of Engineering, Osaka University, Osaka, Japan
Immunology Frontier Research Center, Osaka University, Osaka, Japan
3
Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2

OBJECTIVE
Magnetic resonance imaging (MRI) is a molecular imaging method using nuclear magnetic resonance, and can image
deep tissues in bodies with high spatial resolution. 19F atom has high relative sensitivity to 1H and a 100% isotopic
abundance ratio. In addition, the amount of 19F is very few in body. Thus 19F MRI signals can be monitored withnegligible background signal. 19F MRI probes are suitable for tracking specific cells and imaging enzyme activities inliving animals. We have developed a perfluorocarbon-encapsulated silica nanoparticle, termed FLAME. FLAMEs
show highly sensitive 19F MRI signals, and the surfaces can be modified with various functional groups. However, invivo targeting of FLAME is limited because nanoparticles are often taken up by phogocytes through opsonization. In
this study, we modified the surfaces of FLAMEs with polyethylene glycol (PEG) to reduce the opsonization by forming
a fixed aqueous layer on surface of nanoparticle. We evaluated the blood retention and in vivo distribution of the
FLAMEs by using 19F MR imaging.
METHODSPEG was modified on the surface of FLAME-OH
in one step by silane coupling with PEG-silane. We
compared the blood retention of FLAME-PEG and
FLAME-COOH in living mice by 19F MRI. As
another approach, we also developed a block
copolymer-encapsulated 19F MRI nanoparticle as a
19
F MRI contrast agent. The nanoparticles wereprepared by living radical polymerization on
perfluorocarbon nanoemulsions.
RESULTSTo examine the blood retention of FLAME, the accumulation of FLAME in the liver was evaluated by 19F
MRI. In FLAME-COOH, a strong 19F MRI signal was observed from the liver, while in FLAME-PEG, 19F
MRI signal in the liver is lower.

CONCLUSIONS
By modifying FLAME surface with PEG, we achieved decrease of liver accumulation and improvement of blood
retention.
─ 82 ─

P-17
Visualization of mitochondrial Ca2+ flux in pancreatic INS-1 cells cultured by
the medium with high concentration of fatty acids.
Mariko Suzuki,1 Megumi Kawakubo,1 Naoko Iida-Tanaka2,*

1 Dept. of Health and Nutr. Sci., Grad. Sch. of Stud. in Hum. Cult., Otsuma Women’s Univ.,
2 Dept. of Food Sci., Otsuma Women’s Univ.

OBJECTIVE
Elevated free fatty acid concentrations in plasma is supposed to decline pancreatic β-cell function and mass, resulting inimpaired insulin secretion. The phenomenon, "lipotoxicity", is thought to be responsible for progressive decrease of
insulin secretion in type 2 diabetes. Palmitic acid (PA), one of saturated fatty acids has been reported to induce ER stressand apoptosis in INS-1 cells. Concentration of mitochondrial Ca2+ has been reported to be affected by Ca2+ influx from
ER during ER stress and to modulates mitochondrial metabolism and apoptotic threshold. In this study, we visualized
mitochondrial Ca2+ fluctuation induced by glucose intake, and effects of elevated palmitic acid or oleic acid levels in theculture media on the Ca2+ fluctuation were analyzed.
METHODS
INS-1 cells originated from rat pancreatic β-cells were cultured in the medium with 200 µM of palmitic acid (PA-treatedcells) or oleic acid (OA-treated cells) for 6-7days. For visualization of mitochondrial Ca2+, a fluorescent protein CEPIA
which was Ca2+-binding and mitochondrial targeted protein was used. Cells were transfected with the plasmid encoded
CEPIA cDNA and fluorescent images were obtained by using Olympus IX81 equipped with ORCA-AG CCD camera.
Expression of proteins related to mitochondrial Ca2+ flux, energy metabolism regulation and apoptosis were assessed bymRNA levels obtained from Realtime PCR analyses.
RESULTS
Figure 1 shows that mitochondrial Ca2+ was increased up to ~1.4 fold of the start level 30 min after adding glucose incontrol cells. In the PA-treated cells, in contrast, mitochondrial Ca2+ level was kept at the start level, suggesting that the
number of ATP generated in mitochondria stayed low after addition of glucose. In the OA-treated cells, mitochondrial
Ca2+ level was higher than that in the control cells until ~20 min after glucose addition and was decreased quickly to thelevel ~1.2 fold of the start level.
CONCLUSIONS
A possible hypothesis for those results is that palmitic acids reduce mitochondrial ATP generation and oleic acids,
oppositely, accelerate it. To confirm the hypothesis, further experiments, for example, measurement of ATP, mitochondrial
membrane potential and oxygen consumption rate are necessary.
(B)
Relative fluorescent intensity

(A)

1.8
1.6 
1.4 
1.2
1
0.8
0.6

Control

OA

PA

15

20
25
Timemin

0.4

0

5

10

30

35

40

Fig.1. (A) Fluorescent images of mitochondrial Ca2+ in INS-1 cells transfected with CEPIA2mt and (B) changes of
mitochondrial Ca2+ concentration after adding glucose up to 20 mM in the medium. Intensities of green fluorescence of
CEPIA2mt were measured and plotted as relative values against those at 0 min. PA, the cells cultured in the medium
with 200 µM of palmitic acid; OA, those in the medium with 200 µM of oleic acid.
─ 83 ─

P-18
Anti-apoptotic properties of ferulic acid in pancreatic INS-1 cells.
Sumika Takahashi, Megumi Kawakubo, Naoko Iida-Tanaka*
Dept. of Food Sci., Otsuma Women’s Univ.
OBJECTIVE
Insulin secretion from pancreatic β-cells is strongly affected by various stress, especially oxidation stress and ER stress.
Considerable evidences indicate that oxidative stress induced by elevated saturated fatty acid concentration in plasma
reduces pancreatic β-cell mass in Type 2 diabetes. Ferulic acid is one of polyphenols and rich in rice bran. Ferulic acid has
been reported to have antioxidative and anti-apoptotic effects in pancreatic β-cells. In this study, protective effects of ferulic
acid against "lipotoxicity" of palmitic acid were evaluated by insulin secretion, apoptosis induced by H2O2 and gene
expression of apoptosis- and ER stress- related proteins.
METHODS
INS-1 cells originated from rat pancreatic β-cells were cultured in the medium with 200 µM of palmitic acid (PA) for 67days. Treatment of ferulic acid (FlcA) was performed by adding ferulic acid (final conc. of 0 or 10 µM) to the medium
with PA. Apoptosis induced by H2O2 (final conc. of 1 mM) was evaluated by fluorescent images with Annexin V, andinsulin secretion from cells was visualized with FM1-43 fluorescent reagent. Fluorescent images obtained by using
Olympus IX81 equipped with ORCA-AG CCD camera or Zeiss LSM510 confocal fluorescence microscope. Expressionof proteins related to apoptosis and ER stress was assessed by mRNA levels obtained from Realtime PCR analyses.
RESULTS
Much more apoptosis was induced by H2O2 in the PA-treated cells than in the control cell, suggesting that PA-treatment
lowers apoptotic threshold. Ferulic acid showed an effect reducing the number of apoptotic cells, that is, the effectcanceling toxicity of PA (Fig.1). These results were confirmed by the fact that ferulic acid reduced mRNA level of Ddit3,
increased by PA-treatment. Ddit3 is the protein which is induced by ER stress and promoting apoptosis. In addition, ferulic
acid recovered the early insulin secretion reduced by PA-treatment.
CONCLUSIONS
Ferulic acid has a protective effect against PA-induced toxicities, that is, lowering insulin secretion and inducing apoptosis
in pancreatic β-cells, INS-1. From the analysis of mRNA levels, it was described that Ddit3 which is induced by ER stressand regulates apoptosis is a key protein for lipotoxicity of PA and the protective effect of ferulic acid.
PA

PA + FlcA

5 min after
H2O2 addition

BeforeH2O2 addition

Control

Fig.1. Fluorescent images of H2O2-induced apoptosis visualized by Annexin V.
PA, the cells cultured in the medium with 200 µM of palmitic acid; PA+FlcA, the cells
cultured in the medium with 200 µM of palmitic acid and 10 µM of Ferulic acid.

─ 84 ─

P-19
Cell Morphology and Early-phase Ca2+ Transients of Guinea-Pig Pulmonary Vein
Cardiomyocytes Compared with Atrial and Ventricular Cardiomyocytes
Iyuki Namekata,1 Haruhito Hiiro,1 Tamano Ohmori,1 Yusuke Tanaka,1Shogo Hamaguchi,1 Hikaru Tanaka1
1
Department of Pharmacology, Toho University Faculty of Pharmaceutical Sciences
Pulmonary veins contain a myocardial layer, whose electrical activity is considered to be involved in the genesis
and maintenance of atrial fibrillation. To obtain insight into the automaticity of the pulmonary vein myocardium, westudied the cellular morphology and spatio-temporal pattern of the rise in Ca2+ during the early-phase Ca2+ transientsof isolated guinea pig pulmonary vein cardiomyocytes using confocal microscopy and compared with those ofventricular and atrial cardiomyocytes.
Transsarcolemmal Ca2+ influx during the action potential plateau triggers Ca2+-induced-Ca2+ release (CICR) fromthe sarcoplasmic reticulum. In ventricular cardiomyocytes with a well-developed T-tubular system, Ca2+
concentration rises and rapidly becomes uniform throughout the cytoplasm, while in atrial cardiomyocytes lacking
T-tubules, a wave of CICR propagates from the subsarcolemmal region towards the cell center. The pulmonary vein
cardiomyocytes had cross striations and were rich in sarcoplasmic reticulum but lacked T-tubules; this was similar toatrial cardiomyocytes but different from ventricular cardiomyocytes, in which T-tubules were present throughout thecell. The sarcoplasmic reticulum was present throughout the cytoplasm in all cell types, which was consistent withthe observation that Ca2+ sparks occurred throughout the cytoplasm in all cell types. On the induction of Ca2+transients by electrical field stimulation of the pulmonary vein cardiomyocytes, the rise in Ca2+ concentration firstoccurred at the subsarcolemmal region and then spread to the cell interior; this phenomenon was similar to that in
atrial cardiomyocytes but different from that in ventricular cardiomyocytes, in which the Ca2+ concentration rose
simultaneously throughout the cytoplasm. The properties of the Ca2+ sparks and Ca2+ waves in the pulmonary vein
cardiomyocytes were the same as those in atrial and ventricular cardiomyocytes. Thus, the pattern of the rise in Ca2+
concentration during the early phase Ca2+ transient is not determined by the properties of the Ca2+ release unit itself,
but rather by the presence or absence of the T-tubules.
In pulmonary vein cardiomyocytes, the percentage of cells showing spontaneous Ca2+ transients, as well as theprobability of Ca2+ spark firing, was higher than in the atrial and ventricular myocytes. In the pulmonary vein
cardiomyocytes showing spontaneous activity, the Ca2+ transients were preceded by increased firing of Ca2+ sparks.
SEA0400, an inhibitor of the Na+/Ca2+ exchanger, decreased the frequency of the Ca2+ transients and eventuallyinhibited the Ca2+ transients completely without decreasing the firing of Ca2+ sparks. These results indicate that theguinea-pig pulmonary vein myocardium have a tendency to show spontaneous electrical activity, and that they are
mediated by Ca2+ released from the sarcoplasmic reticulum and the resulting activation of the Na+/Ca2+ exchanger.
In conclusion, in guinea pig pulmonary vein cardiomyocytes that lack T-tubules, Ca2+ transients involve thepropagation of Ca2+ waves, which is similar to that of atrial but not ventricular cardiomyocytes. Ca2+ sparks appear
to be involved in the generation of spontaneous Ca2+ transients of pulmonary vein cardiomyocytes.

─ 85 ─

P-20
Imaging stress-responsive long-distance ROS-Ca2+ signaling in Marchantia
Kenshiro Watanabe,1 Kota Hasegawa1, Hiroki Shindo1, Kenji Hashimoto1,2, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan
Although plants lack the nervous system and brain, as sessile organisms, plants are equipped with numerous sensors
to monitor the environmental changes, signal transmission mechanisms throughout the body as well as decentralized
information processing systems to reorganize their body for environmental adaptation. In response to biotic and abiotic
stresses, long-distance signals are systemically transmitted from the stressed local sites to distant tissues. Suchlong-distance systemic signals play important roles to prevent the entire plant from further damage by stress. Various
signal molecules such as hormones transported through phloem and xylem are involved in slow long-distance signaling.
Moreover, plants can also rapidly transmit signals systemically. Possible involvement of Ca2+ and reactive oxygen
species (ROS) as well as electric signals has been suggested in the rapid long-distance signaling, the molecularmechanisms are largely unknown. We have developed live-imaging systems to monitor cytosolic Ca2+ and ROS in amodel liverwort Marchantia polymorpha and analyzed their dynamics under various stresses.
We established a transgenic plants expressing Ca2+ sensor fluorescent proteins GCaMP6f or R-GECO and monitored
spatiotemporal dynamics of cytosolic Ca2+ concentration. NaCl and wounding treatment induced Ca2+ rise not only at the
local site but also at distant sites and the Ca2+ wave was systemically transmitted throughout the thalli (Fig. 1). The speedof signal transmission was in the range of 1 mm/sec. We also developed the live imaging system of ROS using afluorescent indicator and observed its dynamics. Wound-induced ROS production was observed not only locally but also
at distant sites. These results suggest that the long-distance signaling mechanism involving Ca2+ and ROS exist even in abasal liverwort.
In seed plants, the ROS production by NADPH oxidase is synergistically activated by Ca2+ and phosphorylation[1-4]. The Ca2+-activated ROS-producing NADPH oxidase and ROS-activated Ca2+ permeable channels are postulated
to be co-localized at microdomains in the plasma membrane, and constitute a positive feedback mechanism to amplify
Ca2+ and ROS signals [2-4]. However, molecular identity of Ca2+ channels involved in stress responses remain largely
unknown. By combining the imaging technique and molecular genetic tools, we are trying to identify molecules
involved in the rapid long-distance signaling involving Ca2+ and ROS. Inter-relationship between Ca2+ and ROS signals
in the rapid long-distance signal transmission will be discussed.
1.
2.
3.
4.

Ogasawara et al. (2008) J. Biol. Chem. 283: 8885-8892.
Takeda et al. (2008) Science 319: 1241-1244.
Kurusu et al. (2013) Trends in Plant Science 18: 227-233.
Kaya et al. (2014) Plant Cell 26: 1069-1080.
0 sec (NaCl treatment)

14.5 sec

26.7 sec

NaCl treatment site

Fig. 1 Imaging the propagation of Ca2+ signal in response to NaCl treatment

─ 86 ─

P-21
Regulation of cell proliferation and differentiation by NADPH oxidase-mediated
ROS production in the development of a model liverwort Marchantia polymorphaKazuyuki Kuchitsu1,2, Yuki Hagiwara1, Daisuke Miyamoto1, Kenji Hashimoto1,2
1
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo Univ. of Science, Noda, Japan
Reactive oxygen species (ROS) are highly toxic molecules generated during photosynthesis and aerobic respiration.
However, tightly regulated ROS production by NADPH oxidases/respiratory burst oxidase homologs (Rbohs) integrate
multiple signal transduction network in plants. Rbohs are synergistically activated by Ca2+ binding to the N-terminal
EF-hand motifs and phosphorylation by several families of protein kinases [1-7]. Spatiotemporal pattern of
Rboh-mediated ROS production and the ROS-Ca2+ signal network plays key roles in regulating a broad range ofphysiological processes, such as growth and development including tip growth of root hairs [2,7] and pollen tubes [4],defense responses against biotic and abiotic stresses [3], long-distance signaling as well as cell wall metabolism.
Unlike functional diversification of Rbohs in seed plants (for example, 10 Rbohs in Arabidopsis thaliana [7]), only
two isozymes exist in a model basal liverwort Marchantia polymorpha. Genetic and imaging analyses revealed their
distinct physiological roles in development, morphogenesis and environmental stress responses. MpRbohA is expressedin the cell-dividing apical meristematic zones and its knockout lines showed reduced cell proliferation at the apices aswell as defects in gametophyte development. Moreover, the double mutant mprbohA mprbohB exhibited severe defects
in cell differentiation. These results suggest that Rboh-mediated ROS production is involved in the maintenance of stem
cells as well as the regulation cell proliferation and differentiation. We also performed computer simulation of the growthof the wild type and mprbohA mutant to understand possible roles of MpRbohA-mediated ROS
production. Evolutionary aspects and molecular regulatory mechanisms of Rboh/ROS-mediated signaling will also be
discussed.
1.
2.
3.
4.
5.
6.
7.

Ogasawara et al. (2008) J. Biol. Chem. 283: 8885-8892.
Takeda et al. (2008) Science 319: 1241-1244.
Kurusu et al. (2013) Trends in Plant Science 18: 227-233.
Kaya et al. (2014) Plant Cell 26: 1069-1080.
Kärkönen and Kuchitsu (2015) Phytochemistry 112: 22-32.
Hyodo K et al. (2017) PNAS 114: E1282-E1290.
Kaya H et al. (2019) Plant J. 98: 291-300.

─ 87 ─

P-22
Imaging of ROS dynamics and roles of the ROS-producing NADPH oxidasein tapetal programmed cell death during pollen maturation in rice
Togo Fukunaga1, Jumpei Sawada1, Kazunori Ogawa1, Bunki Toh1, Kenji Hashimoto1,2,
Shigeru Hanamata2,3, Seijiro Ono4, Ken-ichi Nonomura4, Takamitsu Kurusu2.5, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo University of Science,3
Grad. Sch. Sci. & Tech., Niigata Univ., 4Natl. Inst. Genetics, 5Fac. Eng., Suwa Univ. of Science
In flowering plants, programmed cell death (PCD) of the tapetum, the innermost layer of the anther, is one of the mostcritical and sensitive steps for pollen maturation and fertility. It is severely affected by various environmental stresses,
which cause serious problems in agriculture. Production of reactive oxygen species (ROS) has been shown to berequired for post meiotic anther development including the tapetal PCD and pollen maturation in rice. However, these
spatiotemporal dynamics had remained poorly understood.
We here established an in vivo imaging system to analyze the dynamics of ROS in rice anther including thetapetum cells by using several ROS-specific probes during pollen maturation. Imaging analyses showed that ROS
accumulated in the tapetum cells transiently prior to the tapetal PCD. The present monitoring system for ROS
accumulation offers a powerful tool for determining the relationship between ROS signaling and regulation of ricetapetal PCD during pollen maturation.
Furthermore, we identified a ROS producing-NADPH oxidase/respiratory burst oxidase homolog (Rboh)
specifically expressed in rice anther and generated its knockout mutant by genome editing. We will report its phenotype
and discuss possible involvement of NADPH oxidase-dependent ROS production as well as transcriptional network in
the regulation of autophagy and the proper timing of tapetal PCD during pollen maturation.

─ 88 ─

P-23
Imaging autophagy in the anther tapetum cells during pollen maturation in rice
Kazunori Ogawa1, Jumpei Sawada1, Togo Fukunaga1, Bunki Toh1, Kenji Hashimoto1,2,
Shigeru Hanamata2,3, Seijiro Ono4, Kenichi Nonomura4, Takamitsu Kurusu2,5, Kazuyuki Kuchitsu1,21
Dept. Appl. Biol. Sci. & 2Imaging Frontier Center, Tokyo University of Science,3
Grad. Sch. Sci. & Tech., Niigata Univ., 4Natl. Inst. Genetics, 5Fac. Eng., Suwa Univ. of Science
Autophagy contributes to various cellular processes including maintenance of cellular homeostasis, innate immunity,
development, and programmed cell death (PCD) in multicellular organisms. We showed that autophagy is required forpost meiotic anther development including programmed cell death (PCD)-mediated degradation of the tapetum andpollen maturation in rice [1-5]. However, the spatiotemporal dynamics of autophagy in the tapetum remain poorly
understood. We established an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum cells byexpressing green fluorescent protein-tagged AtATG8, a marker for autophagosomes [6]. 3D-imaging analysis revealed
that the number of autophagosomes/autophagy-related structures is extremely low at the tetrad stage, and autophagy isdramatically induced at the uninucleate stages throughout the tapetal cells during anther development. The present
monitoring system for autophagy offers a powerful tool to analyze the regulation of autophagy in rice tapetal cells during
pollen maturation. We will discuss possible mechanisms for the regulation of autophagy and its physiologicalsignificance in tapetal PCD during pollen maturation.
1.
2.
3.
4.
5.
6.

Kurusu et al. (2014) Autophagy 10: 860-870.
Hanamata et al. (2014) Front. Plant Sci. 5: 457.
Kurusu et al. (2016) Bioimages 24: 1-11
Kurusu & Kuchitsu (2017) J. Plant Res. 130: 491-499.
Kurusu et al. (2017) Plant Signal. Behav. 12: e1365211
Hanamata et al. (2019) Plant Biotechnol. in press

─ 89 ─

P-24
Dynamic Photocontrol of Biomachines During Cell Division
Kazuya Matsuo1, Mafy Nusrat Noushaba1, Shota Hiruma2, Ryota Uehara2, Nobuyuki Tamaoki1
1Research Institute for Electronic Science, Hokkaido university
2
Graduate School of Life Science, Hokkaido University
OBJECTIVE
In eukaryotic cell division, chromosomes work as the key player to transfer the genetic information to the next
generation of cells. After chromosome replication in S phase of cell division, the chromosomes migrate from spindle
pole to the metaphase plate during metaphase. In this dynamic process, various biomachines including kinesin anddynein family proteins can concertedly work with high accuracy and precision for the successful cell division. However,the dysfunction of these biomachines can activate the spindle assembly checkpoint through the misalignment ofchromosomes at the metaphase plate. The prolonged mitotic arrest delays the cell-cycle progression, which eventuallyleads to the apoptotic cell death. Thus, “chromosome migration” triggered by biomachines should be the attractive target
for unveiling the mitotic cell division. Herein, we report the novel approach for optochemically controlling thechromosome migration driven by the specific biomachine that is centromere-associated protein E (Cenp-E), using theimaging technique equipped with the light illumination system.
METHODS
Reversibly photoswitchable Cenp-E inhibitor was designed and chemically synthesized by the coupling of theazobenzene derivative with the core structure of the authentic Cenp-E inhibitor, GSK923295. We explored thefundamental properties of the synthesized photoswitchable Cenp-E inhibitor through the photophysical and biochemical
experiments including ATPase assays in vitro and cell-based assays. As the unique technique for controlling the mitotic
cell division, we attempted to establish the light-controllable system of chromosome migration driven by Cenp-E by thealternative illumination of ultraviolet (UV) / visible (Vis) lights.
RESULTS
Our Cenp-E inhibitor exhibited the high photoswitchability based on the reversible cis-trans photoisomeization of theazobenzene moiety by illuminating UV/Vis lights. Before light illumination or upon Vis light illumination, trans isomerin the azobenzene moiety was majorly formed, which worked as the potent Cenp-E inhibitor (IC50 for before light illumination = 5.9µM, IC50 for Vis light = 14 µM from in vitro ATPase assay). In sharp contrast, upon ultraviolet light illumination, thepredominant cis isomer was found to be a non-inhibitor for Cenp-E (IC50 for UV light = 120 µM from in vitro ATPase assay).
Depending on this drastic affinity change of our Cenp-E inhibitor by light stimuli, not only chromosomealignment/misalignement on the metaphase plate but also chromosome migration/positioning in live mitotic cells were
photocontrolled in real time.
CONCLUSIONS
We have successfully developed the photoswitchable Cenp-E inhibitor, which enabled to spatiotemporally regulate
chromosome migration driven by Cenp-E on spindle microtubules using light illumination. The further details will be
discussed in the poster.

─ 90 ─

P-25
Development of Ratiometric Oxygen Probes Based on Green Fluorophore and Red
Phosphor for Visualization of Intracellular Oxygen Level
Mami Yasukagawa, Seiji Tobita, Toshitada Yoshihara*
Department of Chemistry and Chemical Biology, Graduate School of Science and Technology,
Gunma University

OBJECTIVE
Intracellular oxygen is an important indicator for cell metabolism. To clarify the intracellular oxygen level, we haverecently developed ratiometric oxygen probes consisting of an oxygen-sensitive phosphor and an oxygen-insensitive
fluorophore as reference dye [1,2]. In this study, we synthesized new ratiometric oxygen probes NBD-PR7-BTQphen (1)and NBD-PR11-BTQphen (2) (Fig. 1) in which a green fluorescent nitrobenzoxadiazole derivative NBD excitable at 480
nm and a red phosphorescent iridium complex BTQphen are connected with an oligoarginine linker. We investigated thephotophysical properties of 1 and 2 in solution and attempted to visualize the oxygen levels in living cells by using theseprobes.
METHODS
The emission spectra of 1 and 2 in acetonitrile were measured upon excitation at 480 nm. The emission images of
HeLa cells loaded with the probes were observed with a fluorescence microscope equipped with a dual wavelength
simultaneous-imaging system.
RESULTSWe first examined emission spectra of 1 and 2 in acetonitrile upon excitation at 480 nm (Fig. 2). The new ratiometric
oxygen probes showed dual emission bands derived from fluorescence of NBD and phosphorescence of BTQphen.
Under different O2 partial pressures, the fluorescence intensities of the probes were almost constant, whereas thephosphorescence intensities greatly decreased with increasing the O2 partial pressure, indicating that the probes in
acetonitrile are sensitive to O2 concentration.
We next carried out cell culture experiments using the ratiometric probes. The sufficient emission signal could beobtained after HeLa cells was incubated with these probes (2 µM, 2h). Then, we attempted to image the oxygen levels of
HeLa cells using ratiometric probes. when the O2 partial pressure was changed from 21% to 2.5% O2 condition, the ratio
between the phosphorescence and fluorescence intensities increased.
CONCLUSIONS
We designed and synthesized the new ratiometric O2 probes 1 and 2, in which a green fluorescent NBD molecule anda red phosphorescent BTQphen molecule are connected with arginine peptide linker. These ratiometric probes showed
dual emission bands in solutions and living HeLa cells. The O2 levels in living HeLa cells could be imaged by
N saturated
ratiometric measurements using developed probes.
aerated
2

Oligoarginine linker(n = 7, 11)Intensity / arb. unit

1

NBD
(Fluorophore)Fig. 1 Chemical structure of ratiometric oxygen
probesNBD-PR7-BTQphen(1)and
NBD-PR11-BTQphen (2).

Phos.

1

0.5
0
3

Phos.

2

2
1

BTQphen (Phosphor)

Fluo.

0

Fluo.

500

600
700
800
Wavelength / nm

Fig. 2 Emission spectra of 1 and 2 in acetonitrile.

[1] Yoshihara, T.;Yamaguchi, Y.; Hosaka, M.; Takeuchi, T.; Tobita, S. Angew. Chem. Int. Ed., 2012, 124, 4224-4227.
[2] Yoshihara, T.; Murayama, S.; Tobita, S. Sensors, 2015, 15, 13503-13521.
─ 91 ─

P-26
Cell cycle dependence of phototoxicity caused by irradiation of blue laser light*Naohiro Kanamaru1, Mikiya Yamaguchi1, Tetsuya Matsuyama1,
Kenji Wada1, Koichi Okamoto1,
Ai Kawakita2, Kaori Murata2, Kenji Sugimoto2
1
Graduate School of Engineering, Osaka Prefecture University
2
Graduate School of Life and Environmental Sciences, Osaka Prefecture University
INTRODUCTION
Exposure to a large amount of short wavelength light is known to cause diseases such as age-related maculardegeneration (AMD) and retinal photolesion. To prevent diseases, the generation mechanism of phototoxity due to shortwavelength visible light irradiation should be elucidated. We have performed quantitative evaluation of phototoxity
caused by short wavelength light irradiation on living cells by using live cell imaging method. As a result, thedependence of phototoxity on the intensity and wavelength of irradiated light has been partly clarified [1]. In this study,
we investigate the cell cycle dependence of phototoxity caused by the irradiation of blue laser light.
EXPERIMENTAL
Figure 1 shows the experimental setup for live cell imaging using a light emitting diode (LED) and a laser diode (LD).
Nucleus and proliferating cell nuclear antigen (PCNA) of the cell (human malignant melanoma-derived cell:
MDA-MB-435S, culture condition: DMEM, 37℃, 5% CO2) were labeled with fluorescent proteins of mPlum and EGFP,
respectively. The light suitable for the excitation of fluorescent protein was extracted from white LED light by an
excitation filter. Extracted light was reflected with a dichroic mirror and condensed through an objective lens (x60) to
irradiate cells in the laboratory dish. The fluorescence from the cells was detected with an EMCCD camera after passing
through the objective lens, the dichroic mirror and the fluorescence filter. After the laser light was adjusted to a low
intensity with a variable-attenuator, it was guided to the microscope using the surface reflection of a cover glass toirradiate the specific site in the cell.
RESULTSThe center of nucleus of cells in M, G1, middle S and late S phase of cell cycle was irradiated with 405 nm laser light of
100 W/cm2. Table 1 shows the irradiation energy dependence of the cell viability (viable cell number / observed cellnumber) within 24 hours after the laser irradiation to cells in each phase of the cell cycle. As shown in Table 1, the cell
viability decreases with increasing the irradiation energy in all phases of the cell cycle. The viabilities of cells in M andG1 phases are relatively lower than those in middle S and late S phases where DNA is synthesized and repaired.
CONCLUSIONS
To investigate the cell cycle dependence of phototoxity caused by the irradiation of blue laser light, we measured thecell viability within 24 hours after the 405 nm laser irradiation to cells in M, G1, middle S and late S phase. The
viabilities of cells in M and G1 phases were confirmed to be lower than those in middle S and late S phases, suggestingthat the phototoxity of blue laser light on living cells depends on the phase of the cell cycle.
[1] K. Takahashi et. al., The 27th Annual Meeting of the Bioimaging Society of Japan, P-7
Cells
Objective lensFluorescenceDichroic mirror
Fluorescence filter
Lens

Excitation
light

LD
Attenuator
Cover glass

Table 1. The dependence of the phase of the cell
cycle and the irradiation energy on the cell viability
Irradiationenergy（kJ/cm2）

LED (white)
Excitation filter
EMCCD camera

Fig. 1. Configuration of fluorescent microscope─ 92 ─

Cell viability（Viable cell number / Observed cell number）

M

G1

MidS

LateS

0

5/5

5/5

5/5

5/5

12

3/5

2/5

4/5

3/5

18

2/5

1/5

3/5

3/5

24

1/5

0/5

2/5

3/5

Total（%）

20 / 20（100 %）
12 / 20（60 %）
9 / 20（45 %）
6 / 20（30 %）

P-27
Development of novel microbubbles for ultrasound imaging
Tamotsu Maruyama1, Daiki Omata1, Lisa Munakata1, Tadamitsu Shima1, Saori Kageyama1,
Fumiko Hagiwara1, Yuno Suzuki1, Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma Science, Teikyo University
OBJECTIVE
Ultrasound imaging is non-invasive and provide real-time imaging. However, it is difficult to observe a small blood flowsuch as a tumor tissue. In order to solve this problem, microbubbles (MBs) as ultrasound contrast agents are utilized.
Sonazoid which is approved contrast agent is applied for detection of liver and breast cancer. Sonazoid is actively
uptaken into Kupffer cells in liver due to be composed with phosphatidylserine. Therefore, it can negatively stain tumor
region without Kupffer cells in liver. The blood flow imaging in breast cancer is performed at the vascular phase before
liver accumulation of Sonazoid. Generally, the clearance of Sonazoid is fast. Thus, it is thought that the properties of
MBs such as the blood clearance and their stability would be affected by the shell composition of MBs. In this study, we
focused on the shell composition of the MBs and tried to develop MBs with long blood circulation.
METHODS
Liposomes composed of DSPC, DSPG and DSPE-PEG (2k) OMe were prepared using the lipid film hydration method.
The liposome suspension was homogenized (15,000 rpm, 5 min) under the perfluoropropane (C3F8) atmosphere to makeMBs (DSPG-MBs). The DSPG-MBs dispersion was mixed with sucrose solution and freeze-dried. To evaluate blood
circulation of MBs, DSPG-MBs or Sonazoid were intravenously injected into the mice. After injection of MBs, bloodflow in the kidney was observed with the contrast mode of ultrasonography. Next, to evaluate the hepatic accumulationof MBs, DSPG-MBs or Sonazoid were intravenously injected into mice. At 10 minutes after DSPG-MBs or Sonazoid
injection, the mice were perfused to remove DSPG-MBs or Sonazoid in the blood. Then, the liver was extirpated andobserved with contrast mode of ultrasonography. And to evaluate the biodistribution, DiR labeled DSPG-MBs or
Sonazoid were intravenously injected into mice. The mice were observed with in vivo imaging system. The intensity offluorescence in the liver was quantified.
RESULTS AND DISCUSSION
In liposome research, it was reported that addition of DSPG into the lipid membrane could enhance the liposome
stability. Generally, MBs are necessary to escape the reticuloendothelial system (RES) and to be stable in blood flow for
developing the long circulating MBs. Therefore, we assessed the effect of DSPG on circulating time. 60% DSPG
containing-MBs (DSPG60-MBs) showed longer blood circulation than Sonazoid. And the signal intensity of the
DSPG60-MBs in liver was lower than that of Sonazoid. In addition, we also examined the liver accumulation of MBs
labeled with DiR. At 15 min after injection of DiR labeling Sonazoid or DSPG60-MBs, the accumulation of
DSPG60-MBs into liver was lower than that of Sonazoid. These results suggested that DSPG60-MBs were stable inblood flow and long circulation microbubbles by escaping the RES.
CONCLUSION
We succeeded to develop the novel MBs (DSPG-MBs) with long circulation by adding DSPG to the shell compositionof MBs. The hepatic accumulation of DSPG-MBs was lower than that of Sonazoid due to be less uptaken into kupffer
cells. Therefore, DSPG-MBs would be an effective diagnosis agent for many cancers.

─ 93 ─

P-28
Contribution of cellular immune response in tumor growth suppression
by the combination of microbubbles and ultrasoundEri Chiba1, Lisa Munakata1, Daiki Omata1, Tadamitsu Shima1, Saori Kageyama1
Fumiko Hagiwara1, Yuno Suzuki1, Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma Science, Teikyo University
OBJECTIVE
Microbubbles have various behaviors such as oscillation and cavitation under ultrasound field. The oscillation is utilized
to ultrasound imaging of blood flow in microvascular. On the other hand, cavitation would be utilized to drug and genedelivery by opening the transient pore in cell membrane and enhancing the permeability of blood vessels. In addition,
cavitation of microbubbles induces jet stream and/or heat generation. This phenomenon would be applied for direct
killing of cancer cells. Moreover, direct cell damage may be induced the release of tumor associated antigens in tumor
tissue. And this is expected that the combination therapy of microbubbles and ultrasound would activate the cellularimmune response for cancer cells. In this study, we examined the anti-tumor effects by the combination of microbubbles
and ultrasound. Moreover, in this combination therapy, we also assessed the contribution of cellular immune response
for tumor growth suppression.
METHODS
Colon-26 cells (mouse colon carcinoma) were inoculated into the back of mice. After 8 days, our microbubbles wereintratumorally injected and ultrasound (Frequency: 1 MHz, Intensity: 4 W/cm2, Duty: 50%, Burst rate: 2 Hz, Time: 2min) was transdermally exposed toward tumor tissue. The anti-tumor effect was evaluated by measuring tumor volume.
To assess the effect of cellular immunity on tumor growth suppression, we also examined in CD8 positive T cells
depleted mice by injection of anti-CD8 antibody.

CONCLUSION
The combination of microbubbles andultrasound had anti-tumor effect cause of thedirect damage of tumor tissue. This damage
could prime the cellular immune response
which can suppress the tumor growth.
Therefore, this combination therapy would be anovel cancer immunotherapy based onchanging the immunological microenvironment
in tumor tissue.

5000

Non-treatment
US

4000

Tumor volume (mm3)

RESULTS and DISCUSSION
In the combination of microbubbles andultrasound, tumor growth was significantly
suppressed (Fig. 1). And this tumor growth
suppression was cancelled in the CD8 positive
T cell depleted mice. This result suggested thatthe combination of microbubbles andultrasound would be an effective cancer therapy
based on priming the cellular immune response.

BL + US

3000

2000
Treatment

1000

0
0

10

20

30

40

Days after tumor inoculation (Days)

Fig. 1 Tumor growth suppression by the combination of
microbubbles and ultrasound─ 94 ─

P-29
Evaluation of Transporter Function in Brain Microvascular Endothelial Cells Derived
from Human Induced Pluripotent Stem Cells
Toshiki Kurosawa,1 Yuma Tega,1 Tomoko Yamaguchi,3 Tatsuki Mochizuki,2 Yasuhiro Hayashi,1Atsushi Yamashita, 1 Hiroyuki Kusuhara,2 Kenji Kawabata,3 Yoshiharu Deguchi1,*1Fac. Pharm-Sci., Teikyo Univ., 2 Grad.Sch. Pharma-Sci., Univ. Tokyo.,
3
Nat. Instit. Biomed. Innov. Health Nut.
OBJECTIVE
Brain microvascular endothelial cells derived from human induced pluripotent stem cells (hiPS-BMECs) are thought
to have the potential to become an innovative human blood-brain barrier (BBB) model. However, functional expressionof various transporters in these cells has not yet been fully evaluated. This information is important for constructing ahigh-quality BBB model that mimics the in vivo human BBB. The objective of this study was to clarify the expression
and function of nutrient and drug transporters in BMECs derived from hiPS.
METHODS
BMECs were differentiated from the hiPS cell line, IMR90-4. mRNA and protein expression levels of transporters
were measured by the quantitative real-time polymerase chain reaction and immunohistochemical staining analyses,
respectively. Transport function was evaluated by uptake and transcellular transport studies. Drugs were quantified by a
LC-MS/MS system or liquid scintillation counter.
RESULTS
hiPS-BMECs expressed endothelial cell markers, such as platelet endothelial cell adhesion molecule -1, vascularendothelial-cadherin, and glucose transporter 1, and tight-junction proteins such as claudin-5, occludin, and zonula
occludens-1. In addition, cultures of these cells on Transwell membranes showed high TEER values ( > 2000 Ω・cm2),and negligibly small transcellular transport rates of lucifer yellow (a paracellular marker). These results suggest that
hiPS-BMECs are endothelial cells with strong tight junctions.
mRNAs encoding solute carrier transporters, such as high affinity cationic amino acid transporter 1 (CAT1),
sodium-dependent glutamate/aspartate transporter 1 (GLAST), L-type amino acid transporter 1 (LAT1), andmonocarboxylate transporter 1 (MCT1), were highly expressed in hiPS-BMECs. Transcellular transport studies showedthat [3H]L-arginine, [3H]L-glutamate, gabapentin, and [14C]L-lactate (substrates of CAT1, GLAST, LAT1, and MCT1,
respectively) were transported symmetrically and/or asymmetrically across the hiPS-BMEC monolayer. Substrates ofCAT1, GLAST, LAT1, MCT1, organic cation/carnitine transporter 2 (OCTN2), and the proton-coupled organic cation(H+/OC) antiporter, were taken up by hiPS-BMECs time-, temperature-, and concentration-dependently. Furthermore,these uptakes were decreased markedly by inhibitors of the corresponding transporter.
Regarding ABC transporters, breast cancer resistant protein (BCRP), multidrug resistance associated protein (MRP) 1,
MRP4, and MRP5 showed relatively high mRNA and protein expression levels. Dantrolene (a substrate for BCRP)
showed superior asymmetrical transport in the abluminal-to-luminal direction. This asymmetric transport was abolishedby knockdown of the BCRP gene using an electroporation gene transfer method.
CONCLUSIONS
The results of the present study indicate that hiPS-BMECs not only form strong tight junctions, but also express
multiple functional nutrient and drug transporters, including CAT1, GLAST, LAT1, MCT1, the H+/OC antiporter, andBCRP. Our findings contribute to the development of high-fidelity in vitro models of the BBB.

─ 95 ─

P-30
Development of new anticancer drug delivery system with ultrasoundfor cancer therapy

Yuno Suzuki1, Daiki Omata1, Lisa Munakata1, Tadamitsu Shima1, Saori Kageyama1,
Fumiko Hagiwara1, Kazuo Maruyama2, Ryo Suzuki 1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma-Science, Teikyo University
OBJECTIVE
Currently, anticancer drug-encapsulated liposomes are approved for cancer therapy. These liposomal drugs
accumulate to tumor tissue based on the Enhanced Permeability and Retention (EPR) effect. However, most of
liposomal drugs have not provided enough therapeutic effects in many types of cancer. It was reported that apermeability of neo-vasculature in human tumor tissue would not be enhanced due to interstitial pressure in tumor tissue,and existence of pericytes. These obstacles would prevent nano-sized particles such as liposomes from passing across the
blood-tumor barrier (BTB) in human. In addition, to obtain efficient antitumor effects, anticancer drugs should betransferred into tumor cells. Therefore, it is necessary to develop a novel active drug delivery system for tumor tissue.
Previously, we reported that the combination of microbubbles (MBs) and ultrasound (US) could open
blood-brain barrier (BBB). In this BBB opening, the mechanical effect by the oscillation of MBs under ultrasound field
loosen the tight-junction between endothelial cells. In tumor tissue, it is expected that this mechanical effect could induce
BTB opening and enhance liposomal drugs accumulation into the tumor tissue. To achieve the efficient liposomal drugsdelivery, it is important that liposomes are loaded on MBs. Because when liposomes-loaded MBs are exposed to US, thecollapse of MB has two functions such as the release of the liposomes from MB and BTB opening. Finally, thesefunctions would lead liposomal drugs to an active drug delivery for tumor tissue. In this study, we prepared
doxorubicin-encapsulated liposomes (D-Lipo)-loaded microbubbles (D-Lipo-MBs) and assessed the feasibility of
D-Lipo-MB as active drug delivery system.
METHODS
Preparation of liposomes loaded microbubbles
Lipid suspension (DSPC:PEG40-monostearate:DSPE-PEG(2k)-biotin = 90:10:1 in molar ratio) was put into avial. The air in head space of the vial was replaced with perfluorobutane (C4F10) gas, and the vial was capped. Then, thevial was vigorously shaken with VIALMIX® to form MBs. The MBs were incubated with avidins to make avidin
modified MBs (avidin-MBs). To prepare D-Lipo-MB, avidin-MB and biotinylated D-Lipo(DPPC:Cholesterol:DSPE-PEG(2k)-biotin = 55:40:5 in molar ratio) were mixed. To confirm D-Lipo loading on MBs,
the D-Lipo-MB was observed with a fluorescence microscope.
Assessment of cell viability by the treatment of D-Lipo-MB and US
D-Lipo-MBs were added to pancreatic cancer cells (PAN02). Immediately, US (Frequency; 1 MHz, Intensity;
0.5 W/cm2, Exposure time; 10 sec, Duty; 50%) was exposed to the mixture of D-Lipo-MBs and the cells. Afterincubation for 1 day, the cell viability was assessed with MTT assay.
RESULTS and DISCUSSION
D-Lipo-MBs were observed with a fluorescence microscope to confirm whether D-Lipo was loaded on MBs.
doxorubicin-derived fluorescence was detected on the MB surface. This result suggested that D-Lipo was loaded on
MBs by avidin-biotin interaction. In addition, the combination of D-Lipo-MBs and US significantly reduced the cell
viability compared with the treatment of D-Lipo-MBs only. From these results, it was thought that the US triggered
release from MBs was induced and resulted in delivering doxorubicin into cancer cells. Thus, the combination of
D-Lipo-MBs and US would be a useful active delivery system for cancer therapy.

─ 96 ─

P-31
Induction of the accelerated blood clearance (ABC) phenomenon
by polyethyrene glycol (PEG) modified microbubbles
Tadamitsu Shima1, Tamotsu Maruyama1, Takumi Yamada1, Daiki Omata1, Lisa Munakata1,
Saori Kageyama1, Fumiko Hagiwara1, Yuno Suzuki1, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University
OBJECTIVE
Microbubbles (MBs) are clinically utilized as ultrasound contrast agents. In general, MBs have lower frequency ofsevere side effects compared with other contrast agents utilized as other diagnostic methods such as MRI and CT.
Therefore, MBs would be able to apply for repeated use. This point is an advantage of ultrasound imaging with MBs. To
enhance the MB stability and prolong the half-life of MB in blood stream, we have developed the novel type of MBs by
optimization of shell components on MB surface. Our MBs are composed with DSPC, DSPG and PEGylated lipid aslipid shell. Recently, it has been reported that MB modified with PEG induced the accelerated blood clearance (ABC)phenomenon. The ABC phenomenon is normally caused by anti-PEG antibody induction after repeated injection of
PEGylated particles. Therefore, it is thought that our MB might be cleared from blood flow with the repeated injection
by the induction of the ABC phenomenon. The ABC phenomenon may affect the effectiveness of the diagnosis and
therapeutics. Therefore, in this study, we examined whether our MBs induce anti-PEG antibody and the ABC
phenomenon.
METHODS
Liposomes composed of DSPC, DSPG and DSPE-PEG (2k) OMe were prepared using the lipid film hydration
method. The liposome suspension was homogenized (15,000 rpm, 5 min) under the perfluoropropane (C3F8) atmosphereto make MBs (DSPG-MBs). MBs were intravenously injected into mice, and blood was collected at each time point.
The antibody titer of anti-PEG antibody was measured by ELISA. To measure the MB clearance from blood flow, we
observed the renal blood flow after MBs injection with sonography. Seven days later, MBs were re-injected, and renal
blood flow was observed with sonography, and the imaging signals in the contrast mode were measured. Furthermore, toconsume anti-PEG antibody in the mice induced the ABC phenomenon, excess of free PEG was injected. Then, ourMBs were intravenously injected and the cancellation of the ABC phenomenon was observed.
RESULTS and DISCUSSION
The injection of our MBs increased the antibody titer for PEGylated lipids which were the components of MB shell.
In addition, in the re-injection of MBs to mice at 7 days after the first injection, the imaging signal quickly decreased
compared with first injection of MBs. It was suggested that the repeated injection of our MB induced the ABC
phenomenon for our MB. Thus, we assessed the contribution of anti-PEG antibodies on accelerated blood clearance of
our MBs. In this experiment, excess of free PEG and our MBs were intravenously injected into the mice which wasalleviated the ABC phenomenon for our MBs. The shortening of imaging signal was alleviated. This result showed thatanti-PEG antibody was competitively inhibited by free PEG and could not bind to MB. Therefore, it was suggested that
the ABC phenomenon was related to anti-PEG immune response.
Our PEGylated MBs have a property of long circulation after single injection. On the other hand, in the repeatedinjection, they induced anti-PEG immune response. In the liposome research, the induction of the ABC phenomenon
was depending on injection dose and interval. In other MB study, it was reported that anti-PEG antibody was temporally
induced. From these reports, it is thought that there would be some condition to avoid the induction of anti-PEG immune
response and escape of the ABC phenomenon even in the repeated injection. Therefore, we will try to find out theconditions that can escape from the ABC phenomenon in the repeated injection of our MB.

─ 97 ─

P-32
Development and evaluation of antibody-modified nanobubbles
using Fc binding polypeptides for tumor imaging
Nobuhito Hamano1, Yusuke Yano1, Yamato Kikkawa2, Tomomi Kobayashi1, Yoko Endo-Takahashi1,
Ryo Suzuki3, Kazuo Maruyama3, Motoyoshi Nomizu2, and Yoichi Negishi1*
1
Dept. of Drug Deliv. and Mol. Biopharm., School of Pharmacy, Tokyo Univ. of Pharmacy and Life Sci.,
2 Dept. of Clin. Biochem., School of Pharmacy, Tokyo Univ. of Pharmacy and Life Sci.,
3
Lab. of Drug and Gene Deliv. Res., Faculty of Pharma-Sci., Teikyo Univ.
OBJECTIVE
Ultrasound (US) imaging is a widely used imaging technique. The use of US contrast agents such as microbubbles,
which consist of phospholipids and are filled with perfluorocarbon gases, has become an indispensable component ofclinical US imaging, while molecular US imaging has recently attracted significant attention in combination withefficient diagnostics. Microbubbles aid in enhancing the specificity and sensitivity of US imaging and can be applied forvarious types of diseases, particularly tumors. For this purpose, targeting strategies for microbubbles have been
demonstrated. Among these targeting strategies, antibodies have been used as a targeting moiety of microbubbles, andthe avidin–biotin interaction has often been adopted to modify microbubbles with antibodies. However, avidin still has
limitations in its usefulness because of the immunogenicity of streptavidin in humans. Therefore, in this study, as analternative to the avidin–biotin interaction in antibody modification, we developed antibody-modified NBs using
Fc-binding polypeptides for US imaging.
METHODS
We prepared polyethyleneglycol (PEG) -modified liposomes which were composed of DSPC and DSPE-PEG2000. To
modify the Fc-binding polypeptide in the liposomes, we used the post-insertion method. Antibody (anti-CD146
antibody) was added to the polypeptide-modified liposomes and incubated for 15 min at RT. Then, antibody-modified
NBs was prepared by entrapping US imaging gas (C3F8) into antibody-modified liposomes. We examined specificadhesion of antibody-modified NBs to HUVECs by fluorescence microscopy. In US imaging, female KCN nu/nu mice
(5–6 weeks old) were injected s.c. into the right flank region with SKOV3 cells (5 × 106 cells/mouse). We administeredantibody-modified NBs via a tail vein when the tumors reached 100 mm3. US imaging was performed using an Aplio80
US diagnostic machine (Toshiba Medical Systems, Tokyo, Japan) and a 12-MHz wideband transducer with contrast
harmonic imaging at a mechanical index of 0.25.
RESULTSFirst, we prepared anti-CD146 antibody-modified NBs (m146-NBs) using Fc-binding polypeptide. The mean particle
diameter of the non-labeled and m146-NBs ranged from 500 to 700 nm, with a narrow distribution. To examine thespecific attachment of m146-NBs to HUVECs, which are known to express high levels of CD146, the cell attachment of
the NBs was observed by fluorescence microscopy. The m146-NBs exhibited a high level of attachment to the HUVECs,
while attachment of non-modified NBs to the HUVECs was not observed. We next attempted US imaging intumor-bearing mice. The m146-NBs showed strong signals at 1 min post-administration compared to the non-modified
NBs. In addition, the contrast images for the m146-NB group were maintained for up to 20 min, in contrast to theobservations for the non-modified group. This result suggests that m146-NBs may target tumor vessels, leading tolonger US imaging.
CONCLUSIONSIn this study, as an alternative to the avidin–biotin interaction in antibody modification, we developed
antibody-modified NBs using Fc-binding polypeptides for US imaging. m146-NBs displayed specific attachment to
HUVECs. Moreover, US imaging analysis demonstrated that the contrast images for the group injected with m146-NBs
had a longer duration compared with those of the non-modified group. Thus, the modification method using Fc-binding
polypeptides is suitable for providing an antibody linker. Therefore, this antibody-modified method using an Fc-binding
polypeptide may be useful for developing next-generation antibody-modified US imaging agents.

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P-33
CpG oligodeoxynucleotide loaded lipid nanoparticles activate CD8+ T cells
and exert antitumor effect for murine colon carcinoma

Lisa Munakata1, Daiki Omata1, Yasunari Haseda2, Shohei Koyama3,
Yoshiaki Okada4, Yasuo Yoshioka2, 4, Taiki Aoshi2, Ryo Suzuki1, *1Faculty of Pharma-Science, Teikyo University, 2 Research Institute for Microbial Diseases, Osaka University,
3
Graduate School of Medicine, Osaka University
4
Graduate School of Pharmaceutical Sciences, Osaka University
OBJECTIVE
CpG oligodeoxynucleotide (ODN), a ligand of Toll-like receptor 9 (TLR9) is known as adjuvant expect for
activating both of innate and adoptive immune system. Type-A CpG, a kind of CpG ODN has an ability to induce
production of IFN- from plasmacytoid dendritic cells (pDCs) via activation of TLR9. IFN- can activate
Th1-associated immunity. Accordingly, it is anticipated antitumor effect by CD8+ T cells. Nevertheless, CpG ODN has
few efficacy, because of its lower cellular interaction. In this study, we tried to develop novel lipid nanocarrier of CpG
ODN (CpG lipid nanoparticles; CpG LNPs) to overcome this problem. Moreover, we attempted to evaluate antitumor
effect of CpG LNPs and elucidate the contribution of CD8+ T cells in this antitumor effect.
METHODS
Preparation of CpG LNPs; CpG LNPs were prepared with microfluidics (NanoAssemblr Benchtop, Precision
NanoSystems Inc.). Briefly, the lipid solution in ethanol and CpG solution in acetate buffer were mixed in the
microfluidic mixer. This mixture was dialyzed against 5% glucose solution to remove ethanol from CpG LNPsuspension. Further, the CpG LNPs suspension was concentrated by ultrafiltration, then passed through 0.22 m filter.
Evaluation of Antitumor Effect; C57BL/6 mice were inoculated MC38 cells (murine colon carcinoma) in their rightflank. After 9 days from inoculation, CpG LNPs (4.5 g CpG equivalents) were intratumorally administrated. The
administration was repeated 5 times every 2 days. The antitumor effect was evaluated by the tumor volume.
Elucidation the contribution of CD8+ T cells in antitumor effect; To deplete CD8+ T lymphocytes, tumor inoculated mice
were intraperitoneally administrated anti-CD8a antibody at 6 and 13 days after from tumor inoculation. Then they weretreated with CpG LNPs. Finally, at day 21 after from tumor inoculation, tumors were collected and embedded in O.C.T.
compound for making frozen slices. The frozen slices were observed with confocal fluorescence microscope afterstaininig with the following fluorescent dye and antibodies. The nuclei were stained with DAPI, the blood vessels were
stained with anti-mouse CD31-Alexa Fluor 647 and the CD8+ T cells were stained with anti-mouse CD8b-PE.
RESULTS AND CONCLUSION
The average size of CpG LNPs were 54.0 nm and the size distribution was narrow. In the evaluation of
antitumor effect, any tumor growth suppression was not observed in the CpG treated mice. On the other hand, the tumor
growth in the mice treated with CpG LNPs was significantly suppressed. Thus, it was suggested that CpG LNPs could
enhance the antitumor effect but CpG could not its own. Further, we elucidated the contribution of CD8+ T cells to thisantitumor effect. In consequence, the antitumor effect by CpG LNPs were completely canceled in CD8+ T cells depleted
mice. Histological examination of tumor at day 21 from inoculation supported that anti-CD8a antibody administration
depleted CD8+ T cells completely in the tumor tissue. Although MC38 tumor contains many CD8+ T cell infiltrations
without treatment, CD8+ T cell infiltration in the tumor would be more aggressive with CpG LNPs treatment. Therefore,
CpG LNPs would be an immunostimulatory formulation to have an ability to exert antitumor effect via activatingcell-mediated immunity.
ACKNOWLEDGEMENTS
This research was supported by AMED under Grant Number JP19cm0106310.

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P-34
Development of fluorescence / NanoSIMS bioimaging coupled with immunostaining
using novel hybrid probe
Naoya Iwano,1 Wataru Haya,1 Yasuhiko Fujita,1 Kohji Shimoda, 2 Yasushi Miyauchi,3 Takuo Onizuka1
1
Toray Research Center, Inc., 2 Toray Industries, Inc., 3 Kamakura Techno-Science, Inc.
OBJECTIVE
The distribute evaluation of a specific target in a biological sample is one of the most important subject in life sciences.
Recently, there are extensive studies using super-resolution microscope or electron microscope technique which has aspatial resolution beyond the diffraction limit of light, while only few studies have been reported with the high lateral
resolution MS (Mass Spectrometry) imaging. Here, we synthesized a novel probe tag which is compatible both withfluorescence and NanoSIMS (Nano-scale Secondary Ion Mass Spectrometry) analysis, and the probe tag was labelledwith an antibody. We developed the MS imaging method by combining NanoSIMS and an immunostaining method
using the probe-labelled antibody. Our developed MS imaging method has a high lateral resolution (< 50 nm), and wewill show the results obtained by our imaging method.
METHODS
A probe tag detectable by fluorescence (Cy5) and NanoSIMS was synthesized and conjugated to the antibody by amaleimide hinge method. We chose β-Actin in mouse nerve fibers as a model target of the imaging method, andprepared a paraffin-fixed tissue stained by the probe-labelled antibody as a measurement sample. Fluorescence images
with excitation light at 633 nm were observed using a confocal laser microscopy. Subsequently, the surface of the sample
was subjected to NanoSIMS imaging by the Cs+ primary ion beam. The detectors of NanoSIMS were set to various
elements including one derived from the probe tag.

RESULTSThe results of β-Actin imaging in the mouse nerve fiber are shown in Fig. 1 (fluorescence) and Fig. 2 (NanoSIMS).
β-Actin was distributed in periphery of each nerve fiber both in NanoSIMS and fluorescence imaging.

5 μｍ

Fig. 1 Fluorescence imaging

Fig. 2 NanoSIMS imaging

CONCLUSIONSThe new synthesized probe tag can be applicable to fluorescence and NanoSIMS analysis. The NanoSIMS imaging
enables elemental imaging of the sample surface with high lateral resolution (<50 nm), and the result is similar tofluorescence imaging. The developed method combining immunostaining and NanoSIMS has the highest lateral
resolution of MS imaging and can be a powerful tool for bioimaging.
─ 100 ─P-35
Promotion of α2,3-Sialylation of Glycoproteins by a Passport Sequence Tag
Recognized by Cargo Receptor
Rino Yamada1, Hirokazu Yagi1, Rena Honda2,3,5, Maho Yagi-Utsumi1,2,3,5,Kazuhiro Aoki2,4, and Koichi Kato1,2,3,5, *Graduate School of Pharmaceutical Sciences, Nagoya City University, 2 Exploratory Research Center onLife and Living Systems (ExCELLS), 3 Institute for Molecular Science, and 4 National Institute for Basic
Biology, National Institutes of Natural Sciences, 5 School of Physical Science, SOKENDAI (The Graduate
University for Advanced Studies)

1

OBJECTIVE
The 53-kDa ERGIC marker protein, ERGIC-53, and its binding partner, multiple coagulation factor deficiency 2 (MCFD2),
mediate the secretion of blood coagulation factor V (FV) and factor VIII (FVIII) in the early secretory pathway. These two
proteins are known to be the causative gene products of combined deficiency of FV and FVIII, an autosomal recessive
bleeding disorder in which the plasma levels of these coagulation factors are diminished. Cumulative evidence has revealed
that ERGIC-53 possesses a luminal carbohydrate recognition domain that binds the high-mannose-type glycans displayed on
cargo glycoproteins in a pH-dependent manner. Recently, we have identified a 10-aminoacid segment in factor VIII thatis recognized by MCFD2 in the cargo receptor complex. Interestingly, we found that, in addition to secretion level,
α2,3-sialylation level of recombinant erythropoietin was significantly elevated by C-terminal tagging it with the
10-aminoacid segment. This raises the possibility that interactions of the secretory glycoproteins with the cargo
receptor complex not only facilitate their vesicular transport from the ER to the ERGIC but also have impacts on thesubsequent glycosylation pathways in the Golgi apparatus. Herein we address the enhancement mechanisms of
α2,3-sialylation owing to the 10-aminoacid segment tagging through investigations of localization of glycosyltransferase
and secretary routes of tagged glycoproteins.
METHODS
Erythropoietin, a model glycoprotein, with or without the 10-aminoacid segment and α2,3-/α2,6-sialyltransferases fusedwith fluorescence proteins were expressed in a human colon cancer cell line, HCT116, or a human embryonic kidneycell line, Expi293 cells. For analyzing the localization of the fluorescence-labeled proteins, cell images were recorded
using a fluorescence microscope BZ-X700 and BZ-H3XF / Sectioning Module (Keyence).
RESULTSWe found that several trans-Golgi resident α2,3-/α2,6-sialyltransferases were located in distinct subcellular zones. In
addition, the erythropoietin tagged with the 10-aminoacid segment visited the α2,3-sialyltransferase zones on its
secretory route.
CONCLUSIONS
Based on these results, we propose that distinct localizations of sialyltransferases in the trans-Golgi can be determining
factors of specific sialylation of glycoproteins on their secretory pathways. Our findings also suggest that the secretoryroutes of glycoproteins can be switched just by tagging them with the specific aminoacid segment, offering a novel
strategy for controlling glycoforms of recombinant glycoproteins of biopharmaceutical interest.

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P-36
Quantitative analysis of cell cycle progression within heterogeneous cell population
using information theory
Ryu Yamanaka,1, 2 Kotaro Oka1, 3, 4, *Dep. of Biosci. and Info., Fac. of Sci. and Tech., Keio Univ., Japan, 2Faculty of Pharm. Sci., Sanyo-Onoda
City Univ., Japan, 3Grad. Inst. of Med., Col. of Med., Kaohsiung Med. Univ., Taiwan, 4Waseda Res. Inst. for
Sci. and Eng., Waseda Univ., Japan. *Corr. Auth.
1

OBJECTIVE
Biological systems utilize even variability and heterogeneity within population for their efficientsustainability and survival at all levels from cells to ecosystems. The properties of individual cells andcollective features of cell population influence each other, and the interaction between the part and wholeacross levels of organization orchestrates the dynamical changes of cellular states including differentiationand cell cycle progression. Each cell continuously replicates identical copies with the allowance of somefluctuations, and it enables the process of the evolution in cell population. Thus, cell proliferation is afundamental process for not only survival of organism but also evolution of living system. However, it has
been little known what to extent cells adjust to proliferation and what type of sub-populations proliferates
quickly in multi-cellular populations.
METHODS
Massive-scale data of signal dynamics from cell-cycle reporter Fucci2 at a single-cell level in HeLa
cells were obtained by the combination of fluorescence live cell imaging and image-processing-based celltracking algorithm. Shannon’s information theory was applied for quantification of the properties ofindividual cells within heterogeneous population during cell cycle progression, which has temporally
different probabilistic distributions, i.e. time-variant system.
RESULTS
Our analysis revealed that cells proliferate with keeping the characteristic levels of variation ateach stage of cell cycle, and cells with the high rate of proliferation likely to belong to the minority population.
This suggests that cells are not completely adjusted to the proliferation. Furthermore, cells growing fast
during cell cycle progression are likely to pass the major transition at each step, and change into the cellstates with more choice at the next step. Together, cells growing fast are likely to progress through cell cyclewith more frequent changes at short-term transition and with less frequent trajectory in long-term
vicissitude.
CONCLUSIONS
Our approach on the basis of information theory revealed that the progression of cell cycle is notglobally but locally optimized in HeLa cells. These findings possibly show the rules that individual cancer
cells within heterogeneous population are subjected to in the process of tumor occurrence and development.
Thus, our observation about the part-whole interaction cross the levels of organization gives the novel
insights to both biology and medicine.

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P-37
Simultaneous visualization of intracellular energy substrates
by using dual FRET imaging
1

Yutaka Shindo,1 Mitsuru Iizumi,1 Ryu Yamanaka,2 Kohji Hotta,1 Kotaro Oka1,3,4,*
Biosci Info., Keio Univ., 2 Pharmac. Sci. Sanyo-Onoda City Univ., 3 Inst. Med., Kaohsiung Med. Univ.,
4
Waseda Res Inst. Sci., Waseda Univ.

OBJECTIVE (11pt)
Mammalian cells consume glucose and produce ATP in two pathways. One is glycolysis, which yields ATP andpyruvate. The other is oxidative phosphorylation in mitochondria. A portion of pyruvate is utilized in mitochondria as anenergy substrate. And the other portion of it is converted to lactate, which is also utilized energy substrate or released to
extracellular fluid. Balance between glycolysis and oxidative phosphorylation is varied in different cell types. For
example, astrocytes dominantly use glycolysis and yield and release lactate, while neurons mainly use oxidative
phosphorylation by using pyruvate and received lactate from astrocyte as substrates. Therefore, to understand energy
metabolism in cells, measuring these two energy substrates is important. In this study, we aimed to develop different
color version of genetically encoded lactate or pyruvate sensor and to achieve simultaneous visualization of lactate andpyruvate by using dual FRET imaging technique.
METHODS (11pt)
Different color version of lactate or pyruvate sensor were developed from genetically encoded FRET based lactate
sensor, Laconic (Martin et al., 201), or pyruvate sensor, Pyronic (Martin et al., 2014), by changing fluorescent proteinsfrom mTFP and Venus pair to the other colored pairs. The developed sensors were induced in HeLa cells, cultured rat
hippocampal neurons and cultured rat astrocytes, and dual FRET imaging was performed on confocal microscope.
RESULTS (11pt)
In the developed color-converted sensors, only red-colored Pyronic (LSSmOrange and mKate2 pair) responded to its
substrate. LSSmOrange is a long stokes shift orange fluorescent protein, and we can simultaneously excite it and mTFP
at 440 nm. Therefore, by using original Laconic (mTFP and Venus pair) and red-shifted Pyronic, one-excitation andfour-emission type dual FRET imaging was achieved. Upon an application of pyruvate (1 mM), cytosolic pyruvate
concentration increased steeply followed by a gradual increase in lactate concentration. On the other hand, an applicationof lactate (1 mM) increased only cytosolic lactate concentration and had no effect on pyruvate concentration. Glutamate(100 M), an excitatory neurotransmitter, increased both lactate and pyruvate concentration in cultured neurons, whichsuggesting an upregulation of glycolysis. It also induced decrease in cytosolic ATP concentration measured by using
genetically encoded ATP sensor, ATeam (Imamura et al., 2009). The glutamate-induced decrease in cytosolic ATPconcentration was attenuated in lactate (5 mM) containing medium. It indicates that lactate was taken into neurons andutilized as energy substrate in exciting neurons.
CONCLUSIONS (11pt)
Simultaneous imaging of intracellular lactate and pyruvate by using dual FRET imaging was achieved by developing
red version of pyruvate sensor, pyronic (LSSmOrange and mKate2 pair). It enabled us to compare the difference inenergy metabolism in various cell types.

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P-38
Intra-vital fluorescence imaging of interaction between transplanted stem cells andimmune cells by quantum nano-materials and tissue clearing (CUBIC) technology
Toshiki Mizumaki,1,* Hiroshi Yukawa,1,2,3 Kodai Kitamura,1 Daisuke Onoshima, 1,2 Etsuo A. Susaki, 4,5
Hiroki R. Ueda, 4,5 Yoshinobu Baba, 1,2,3,6
1
Dept. Biomolecular Eng., Grad. Sch. Eng., Nagoya Univ., 2 Inst. Nano-Life-Systems, Inst. Innovation for
Future Society, Nagoya Univ., 3National Institute of Radiological Science (NIRS), National Institute forQuantum and Radiological Science and Technology (QST), 4Dept. Systems Pharmacology, Grad. Sch. Med.,Tokyo Univ., 5Laboratory for Synthetic Biology, Center for Biosystems Dynamics Research, RIKEN, 6HealthResearch Institute, AISTOBJECTIVE
In regenerative medicine, cell transplantation therapy plays an extremely important role in the diseases that are still
difficult to treat by tissue and organ reconstruction. It is absolutely essential to elucidate the interaction between
transplanted cells and immune cells for the realization and further development of cell transplantation therapy. In thisstudy, we worked on establishing an intra-vital imaging technique using two-photon excitation microscope to observe
the real-time behavior of immune cells against stem cells transplanted to acute liver failure model mice. Furthermore, we
tried to elucidate the comprehensive interaction between transplanted stem cells and immune cells in the liver byapplying tissue clearing technology.
METHODS
Adipose tissue-derived stem cells (ASCs) were labeled with quantum dots (QDs), a fluorescent nanoparticle which has
optical properties of high definition, high sensitivity and long fluorescence lifetime, by introducing QDs into ASCs using cell
membrane penetrating peptide. After labeling with QDs, ASCs were transplanted to acute liver failure model mice fromthe tail vein. At first, time points at which transplanted ASCs began to be eliminated were examined by observing fixedtissues at some time points of 1 to 72 hours after ASCs transplantation. Then, intra-vital fluorescence imaging ofinteraction between ASCs and immune cells was performed at appropriate time point by incising abdomen of mouseunder anesthesia administration and pressing the lens of two-photon excitation microscope against liver that transplanted
ASCs were accumulated. Next, the liver was cleared by tissue clearing technology called CUBIC (Clear, Unobstructed
Brain/Body Imaging Cocktails and Computational analysis), and comprehensive imaging of cell-to-cell interaction was
examined using Light sheet fluorescence microscopy.
RESULTSAs a result of intra-vital fluorescence imaging of interaction between transplanted stem cells and immune cells by
two-photon excitation microscope, we succeeded in capturing the moment at which immune cell approaches totransplanted stem cell for phagocytosis. Furthermore, we succeeded in comprehensively visualizing the function of
immune cells against transplanted stem cells in the inflamed tissue by clearing liver of acute liver failure model mice.
CONCLUSIONSThe present study enabled us to establish an intra-vital fluorescence imaging technique to observe the real-time
interaction between transplanted stem cells and immune cells in inflamed liver. Tissue clearing is expected to be a very
effective method for comprehensive visualization of the function of immune cells against transplanted stem cells.

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P-39
Brain-targeted Gene Delivery using Microbubbles and UltrasoundDaiki Omata1, Fumiko Hagiwara1, Lisa Munakata1, Tadamitsu Shima1,
Saori Kageyama1, Yuno Suzuki1, Kazuo Maruyama2, Ryo Suzuki1,*
1
Laboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University
2
Laboratory of Theranostics, Faculty of Pharma-Science, Teikyo University
OBJECTIVE
The blood brain barrier (BBB) restricts transportation of molecules between the blood and the brain parenchyma. The
BBB is a major obstacle to development of effective medicine for treatment of brain diseases, as many drugs including
macromolecules cannot pass the BBB and reach the brain parenchyma. Recently, it has been noted that gene therapy
could be useful to treat brain diseases, such as Alzheimer’s disease, Perkinson’s diseases and Huntington’s disease. To
achieve successful therapy, a development of gene delivery systems which are safe and efficiently can deliver genes aredesired. A combination of ultrasound (US) and microbubbles (MBs), which is US contrast agent, has attracted attention
as promising technology for brain-targeted drug delivery. We previously reported that the combination of MBs and US
could be useful for delivering genes in vitro and in vivo. In this study, we examined the feasibility of MBs and US forbrain-targeted gene delivery.
METHODS
Mixture of MBs (500 µg of lipid) and pcDNA3-Luc (100 µg), which is a plasmid DNA (pDNA) encoding the firefly
luciferase gene under the control of cytomegalovirus promoter, was injected into tail vein of ddY mice. Immediately,
brain was exposed to transcranial non-focused US (Frequency: 1 MHz, Intensity: 1.2 W/cm2, Duty: 10%, Time: 1 min).
After 1 day, luciferase activities in the brain and several tissues were measured by a luciferase assay system. To
determine in which cells gene expression was induced, immunofluorescence analysis was performed. Briefly,
Venus/pCS2, which is an expression vector encoding mutant GFP gene, was delivered to the brain using MBs and US.
After 2 days, the brain was collected and cryosections were prepared. The sections were stained with antibody against
GFP, followed by treatment of tyramide signal amplification system. In addition, the sections were stained with antibody
against CD31, which is a marker of endothelial cells, and fluorescent secondary antibody. The cells expressing GFP was
observed using confocal laser scanning microscopy.
RESULTS
The treatment of MBs and US showed about 1000-hold higher luciferase activity in the brain than that of treatment with
only pDNA, pDNA with US, or pDNA with MBs. To assess the brain selective gene delivery, we examined gene
expression in several tissues after treatment of MBs and US. As a result, luciferase activity of the brain treated with MBs
and US is higher than that of other tissues such as liver, kidney, heart, lung and spleen. For evaluation of the damage tobrain, we observed the appearance of brain treated with MBs and US. Neither hemorrhage or degeneration of brain wasobserved. These results suggested that MBs and US could selectively deliver genes into brain without notable damage.
As a result of immunostaining, GFP expression was observed in not only endothelial cells but also parenchyma cells.
This result suggested that MBs and US could deliver genes to parenchyma cells through the BBB.
CONCLUSION
MBs and US selectively delivered genes into brain without notable damage and gene expression in parenchymal cells
was observed. In conclusion, it is expected that the combination of MBs and US would be a useful tool to establish
minimally invasive brain-targeted gene delivery system.

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P-40
Blood-brain barrier opening using 220-kHz transcranial MRI-guided focusedultrasound and microbubbles in mouse and rat
1Michiharu Yoshida, 2*Kazuo Maruyama, 3 Yasutaka Kato, 4Rachmilevitch Itay, 4Shuji Suzuki, 4Omer

Brockman, 4Yeruham Shapira, 5Kenji Moriyama, 2Daiki Omata, 2Ryo Suzuki, 1Hajime Kamada
1
Department of Neurosurgery, Hokuto Hospital, 2Faculty of Pharma-Sci., Teikyo Univ., 3Laboratory ofCancer Med-Sci., Hokuto Hospital, 4InSightec Ltd., 5Department of Radiology, Hokuto Hospital

OBJECTIVE
In neuro-oncology, it is believed that one major obstacle to effective chemotherapy is the high vascularity andheterogenous permeability of brain tumors. Focused ultrasound (FUS) exposure with the microbubbles has been shown
to transiently open the blood-brain barrier (BBB) without depositing thermal energy, and thus may enhance the deliveryof various therapeutic drugs into brain tumors. The aim of this study was to evaluate the BBB opening using 220-kHz
transcranial MRI-guided FUS (TcMRgFUS) device and microbubbles in mouse and rat.
METHODS
The experiments were performed with the 220-kHz ExAblate Neuro TcMRgFUS system (InSightec) and novel lipid
bubbles (LB, Teikyo Univ.). Normal mouse and rat brains were irradiated with TcMRgFUS (output power, 5W; durationof irradiation, 30 s; duty cycle 100%) following intravenous injection of 6×107 LB per mouse and rat, respectively. On
irradiation, target temperature rise was monitored by MR thermometry. Immediately after irradiation, BBB opening andcomplications were detected based on T1, T2, T2*, and Gadolinium (Gd) enhanced T1-weighted images.
RESULTS
The maximum temperature of brain tissue was under 42 ℃. The FUS-LB exposure induced successful BBB opening
effect in both mouse and rat, confirmed by Gd enhancement in the target region, lateral ventricles, and sulcus (Figure). In
addition, there were no complications such as edema, coagulation, and hemorrhage.
CONCLUSIONS
Although there remain many conditions to be optimized, BBB opening using a 220-kHz TcMRgFUS device and LB
can offer a non-invasive and feasible drug delivery for brain malignancies.

─ 106 ─P-41
Enhanced chemotherapy effects on gynecological cancers by using the combination oflipid bubble and ultrasoundKohei㻌 Yamaguchi1, Yoko Matsumoto1 *, Haruka Nishida1, Daiki Omata2, Michihiro Tanikawa1,
Kenbun Sone1, Katsutoshi Oda1, Yutaka Osuga1, Ryo Suzuki2, Kazuo Maruyama2, Tomoyuki Fujii1
1
The Department of Obstetrics and Gynecology, The University of Tokyo
2
Laboratory of Drug and Gene Delivery System, Faculty of Pharma-Science , Teikyo University
OBJECTIVE
Delivering anticancer drugs effectively to tumor cells with less side effects, is a key strategy in cancer treatment. Lipid
bubble (LB) is a compound which increases vascular permeability of the tumor under diagnostic ultrasound (US)exposure and enables drugs to be carried effectively to the tumor cells. The aim of our study is to establish a novel drugdelivery strategy for chemotherapy, by using LB and the US exposure, in gynecological cancers.
METHODSAll studies were approved by our institutional animal care and use committee. We developed endometrial cancer andcervical cancer cell lines in vitro. Then we administrated cisplatin or doxorubicin with or without LB under theultrasound. The anticancer effects were evaluated by MTT assay.
We made xenograft models with nude mice. LB with chemotherapy was injected intravenously and the US was applied
onto the tumor. The anticancer effect was evaluated by the tumor size and TUNEL staining. Drug penetration andaccumulation were analyzed by laser-scanning microscope.
RESULTS
When administrated with LB and US exposure to cancer cells in vitro, the viability of both cervical and endometrial
cancer was not reduced, compared with chemotherapy alone. But in vivo models, Tumor volume reduction by either
cisplatin or doxorubicin was enhanced by the administration of LB and US exposure. Drug accumulation by this drugdelivery strategy was confirmed in laser-scanning microscope.
CONCLUSIONSThe combination of LB with US enhanced the effect of anticancer drugs. This technology may be a promising drugdelivery system in the clinical settings.

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P-42
Bioimaging of inhalable nanocarriers with aggregation-caused quenching dye
to visualize retention in the respiratory system
Kohei Yamada,1 Tetsuya Matsushita,1 Kurt D. Ristroph, 2 Hoang D. Lu, 2 Yoshiki Seto, 1 Wei Wu, 3
Hak-Kim Chan, 4 Robert K. Prud’homme, 4 Hideyuki Sato, 1 Satomi Onoue 1,*
1
Laboratory of Biopharmacy, School of Pharmaceutical Sciences, University of Shizuoka, Japan,
2
Department of Chemical & Biological Engineering, Princeton University, USA,
3
Key Laboratory of Smart Drug Delivery of MOE & PLA, Fudan University, China
4
Advanced Drug Delivery Group, The University of Sydney, Australia
OBJECTIVE
Retention of inhaled drugs in the respiratory system is desirable for effective treatment of lung and respiratory diseases.
However, inhaled drugs are rapidly cleared toward the pharynx by mucociliary clearance (MCC), an innate clearance
mechanism of the respiratory tract. To develop inhalable nanocarriers (NCs) with excellent pulmonary retention,
controlling the diffusiveness of NCs in airway
mucus upon mucosal drug delivery system could be
a promising strategy. This study was undertaken toevaluate retention of mucopenetrating andmucoadhesive NCs upon fluorescence imaging with
P2 probe, a self-developed environment-responsive
aza-BODIPY dye quenching in water.
METHODS
Aggregation-caused quenching effect and rekindling of P2 probe were evaluated in simulated lung fluid (SLF). NCs
containing P2 probe were prepared by flash nanoprecipitation and coated with neutral polymer (NCs/N) for
mucopenetration and with cationic polymer (NCs/C) for mucoadhesion. Physicochemical properties of prepared NCs
were characterized. After intratracheal administration of NCs to rats, the lungs were removed and subjected to imaging
by in vivo imaging system (IVIS). Sectioned lungs were observed with confocal laser scanning microscope (CLSM).
RESULTSThe fluorescent intensity of P2 in acetonitrile solution abruptly decreased when the ratio of SLF to acetonitrile reached50%. While quenched P2 exhibited rekindling in acetonitrile and 1%-Tween 80 solution, marked rekindling was notobserved in SLF. These properties of P2 suggest its applicability as an environment-responsive dye for reliablefluorescence imaging of insufflated NCs. P2-loaded NCs/N and NCs/C had a mean diameter of approximately 150 nm
and high dispersion stability in SLF up to 6 h. NCs/N and NCs/C possessed -potentials of approximately 4 and 40
mV, respectively. Although the fluorescent intensity of both NCs in aqueous solvent slightly decreased in 4 h, theleakage of P2 from NCs was limited. Quartz crystal microbalance analysis showed that NCs/C strongly interacted withmucin, a major component in mucus, compared with NCs/N. NCs/N, which had limited interactions with mucin,
exhibited high mucodiffusiveness compared with NCs/C in a diffusion test using artificial mucus. CLSM images ofthe lungs excised at 30 min after intratracheal administration in rats showed that NCs/C were present in clumps awayfrom the epithelium. On the other hand, NCs/N displayed homogenious distribution across the respiratory surfaces inclose proximity to the epithelial cells, suggesting rapid mucus penetration. Intratracheal NCs/C were cleared fromlower RT to the pharynx, possibly due to MCC. In contrast, intratracheal NCs/N exhibited better pulmonary retention,
which may be due to their avoiding MCC because of their high mucodiffusiveness in the respiratory system.
CONCLUSIONS
Mucopenetrating nanoparticulate drug delivery would be a promising approach to offer prolonged drug exposure to therespiratory system, possibly resulting in better clinical outcomes.
─ 108 ─P-43
Development of Caged Gene Regulators forApplication to Control of Genetic Recombination in vivo
Kurumi Yoshida,1,* Masafumi Minoshima,1 Kazuya Kikuchi 1,2
1
Graduate School of Engineering, Osaka University. 2 Immunology Frontier Research Center, Osaka Univ.
OBJECTIVE
Gene targeting through site-specific homologous recombination is a powerful tool for generating predetermined changes
in genomes and controlling gene expression in vivo. Cre-ERT2/loxP system, a modified Cre/loxP system, allows
homologous recombination induced by exogenous chemical ligands. Cre-ERT2, a fusion protein of Cre recombinase with
mutated ligand-binding domain of human estrogen receptor (ER-LBD), does not bind endogenous 17-estradiol (E2) but
binds synthetic ER ligands such as 4-hydroxycyclofen (4-OHC). While this system enables ligand-dependent genetic
recombination, the precise spatiotemporal control is limited due to the diffusion of the ER ligands after administration.
Thus, we focused on photoresponsive caged ER ligands for spatiotemporal control of gene expression using CreERT2/loxP system. Moreover, two-photon illumination enables three-dimensional control of cells within complex anddeeper tissue. However, current caged ER ligands have small two-photon uncaging action cross section, which limits toapplications in deeper tissues using two-photon irradiation. In this study, we aimed to develop caged ER ligands with large
two-photon uncaging action cross sections to control gene expression in vivo.
METHODSWe designed and synthesized caged 4-OHC incorporating nitrobiphenyl groups as photoremovable protecting groups(PPGs) with large two-photon uncaging action cross sections. Photocleavage reaction was initially performed by onephoton excitation using UV light. We analyzed the reaction of the caged 4-OHC by high-performance liquid
chromatography and mass spectrometry. Moreover, we investigated whether light irradiation would induce genetic
recombination through Cre-ERT2/loxP system in caged 4-OHC-treated cells.
RESULTSWe developed caged 4-OHC (ANBPC1 and ANBPC2) incorporating 2-(4’-(bis(methyl)amino)-4-nitro-[1,1’-biphenyl]3)yl)propan-1-ol and 2-(4’-(bis((2-methoxyethoxy)ethyl)amino)-4-nitro-[1,1’-biphenyl]-3)yl)propan-1-ol, respectively.
HPLC analysis indicated that 4-OHC was released in a time-dependent manner after light irradiation. Furthermore, using
reporter genes, we observed robust genetic recombination in ANBPC2-treated cells after light irradiation.

CONCLUSIONSIn summary, we developed caged 4-OHC to photocontrol genetic recombination by incorporating nitrobiphenyl groups
with large two-photon uncaging action cross sections. We found induction of reporter gene expression with ANBPC2 andlight, which demonstrates light-controlled homologous recombination through Cre-ERT2/loxP system in living cells. Our
future effort will focus on precise spatiotempotal control genetic recombination in vivo using caged 4-OHC and twophoton illumination.
─ 109 ─P-44
Image evaluation of biological tissue using Bio-LSI
Ryoma Kumagai,1 Ankush Prasad,2 Teruya Sato,1 Kosuke Ino,3 Hitoshi Shiku,3Shigenobu Kasai1(1Graduate Department of Electronic, Tohoku Institute of Technology, Sendai, Japan, 2Department ofBiophysics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,
Palacký University, Olomouc, Czech Republic3Graduate School Department of Applied chemical –electrochemical science and technology, Tohoku University, Sendai, Japan)

1. OBJECTIVE
Scanning electrochemical microscopy (SECM) is a method that can be employed for imaging tissues using
amperometry. There is a problem which limit is wide usage that it takes longer scanning time. Therefore, Bio-LSI, which
is an electrochemical device capable of measuring 400 electrodes simultaneously, was developed. We have evaluated the
distribution of POD activity in plant tissues using SECM1) and the photosynthetic activity of spinach young leaves using
Bio-LSI2). In this study, we focused on the evaluation of biological tissue and carried out real-time imaging evaluation ofperoxidase (POD), a growth factor of plant tissue using Bio-LSI, and respiratory activity in human epidermal cells.
2. METHODS
1) Real-time imaging of plant tissue POD activity
We used ferrocene methanol (FCCH2OH) as an electron transfer mediator and evaluated POD activity by the reduction
current of FC+CH2OH. We made a cruciform cut on the celery leaves. We added 0.5 mM FCCH2OH and 0.05 mM
H2O2 mixed solution to Bio-LSI, added Ag/AgCl reference electrode, counter electrode (Pt wire (2.4 mm2)), andapplied +0.05 Vvs.Ag/AgCl. We fixed celery leaves on a Bio-LSI sensor and grasped real-time images.
2) Respiratory burst imaging in epithelial cell sheet
We performed simultaneous measurement of respiratory activity of a cell sheet in UV-irradiated area and non-irradiatedarea by irradiating UV after covering the cell sheet with a mask which has a cruciform light transmittable area. We put 2
mL of 11.4 mM glucose-containing phosphate buffer solution for measurement into the Bio-LSI and placed an Ag/AgCl
reference electrode and a counter electrode (Pt wire). We filled the chamber with oxygen gas and applied -0.5
Vvs.Ag/AgCl. We then excised a cell sheet and put a mask, irradiated UVB to irradiation energy of 1.5 J/cm2, introduced
the cell sheet at a distance of 100 um between the electrode and the cell sheet and performed the measurement.
3. RESULTS AND DISCUSSION-400 pA
1) Real-time imaging of plant tissue POD activity
Fig.1(A) shows a photograph of damaged celery leaves.
Figs.1(B and C) show the POD activity in real-time of(C)
1 mm (B)-100 pA
celery leaves. These images reflect the cruciform (A)Fig.1 The celery leaf photography and real time image
scratches of celery leaves.
(A) Injured celery leaf photograph2) Respiratory burst imaging in epithelial cell (B), (C) Real-time image after 90 seconds after 120 seconds
from the start of POD activity measurement
sheet
cell sheet(-)
Fig.2(D) shows a photograph of a cell sheet irradiated-8 nA
mask(+)
with UV and Fig.2(E) shows a bio-LSI image of the cellsheet. Fig.2(E) shows that the oxygen reduction current
value in the irradiated area is the lowest. This result showsthat this area is causing a respiratory burst. We alsomask(-)
0 nA
(E)
(D)
obtained respiratory activity images in areas with theother cell sheet.
Fig.2 Picture of cell sheet and respiratory activityof cell sheet by UV stimulation
4. CONCLUSIONS(D) Picture of cell sheet
In this study, we have captured real-time images of(E) Image from the installation after 5 minutes
celery's POD activity and image of a cell sheet normal
respiration and respiratory bursts caused by UV stimulation. In the future, we plan to focus on the simultaneous imaging
of POD activity and the production of Reactive Oxygen Species (ROS) in plant tissue.
REFERENCES(1)H. Zhou, et al., Bioelectrochemistry, 2001, 54, 151-156., (2) S. Kasai, et al., 231st ECS MEETING, 2017, MA2017-01 1732.
─ 110 ─

P-45
Study on Photodynamic diagnosis for micrometastases using photosensitizer
Talaporfin sodium
1Saito Takuma1 , 2Furuya Kazunari1 , Li Liming1

Graduate School of Chitose Institute of Science and Technology

1

OBJECTIVE
The first cause of death in Japanese is cancer and the first ranking of females is colon cancer. In Japanese after colorectal
cancer treatment, the site of resection where the cancer cells first occur and lymph node dissection are important toprevent metastasis and prolong the patient's life. In the current medical field, it takes time to investigate the metastasis ofdissected lymph nodes. Therefore, the method of photodynamic diagnosis (PDD) real time diagnosis was examined.
PDD is a diagnostic method that accumulates a photosensitizer having affinity for cancer cells at the cancer site of apatient and observes fluorescence obtained by excitation of light of a specific wavelength. In this study, we are aimed todetect colon cancer lymph nodes and evaluate metastasis, based on the fluorescence spectra and images derived fromphotosensitizers.
METHODS
Human colon adenocarcinoma cell line HT29 was incubated with Talaporfin for 1 hour. Thereafter, the medium was
replaced with fresh medium, cultured for 8 hours or 12 hours, and measured by FACS. HT29 was used to create mouse
lymph node metastasis model. Talaporfin was administered to the tail vein at the dose of 10 mg / kg after 6 or 12 hours
before the experiment and Indocyanine Green (ICG) was administered at the dose of 5 mg / kg near the anus to themouse model. Three samples were used for each mouse under each condition. After sacrificing the mouse, visual images,
fluorescence images and fluorescence spectra were measured using a laparoscopic system for lymph node metastasis.
Lasers with wavelengths of 405 nm and 664 nm to excite Talaporfin and 785 nm to excite ICG were used as theexcitation light sources.
RESULTSAs a results of in vitro, HT29 retained Talaporfin even after culture for 8 or 12 hours. Fig.1 is a fluorescence image andan HE-stained tissue image in a lymph node. Fluorescence of Talaporfin accumulating in cancer at the lymph node andfluorescence of ICG capable of detecting lymph nodes were obtained from the same position. Therefore, colon cancer isthought to have spread to lymph nodes. Metastasis of cancer was confirmed from HE stained tissue image. Fig.1 (F) isthe fluorescence spectrum in the lymph node. In cancer metastatic lymph nodes, Talaporfin fluorescence was obtained in
the vicinity of 670 nm for 12 hours after.
CONCLUSIONS
Based on the above results, Talaporfin was accumulated in cancer metastatic lymph nodes. We demonstrated thepossibility of diagnosis of colon cancer lymph node metastasis by using Talaporfin.

Fig.1 mouse model imaging & fluorescence spectrum: (A) White light + visible image (white arrow is tumor GFP), (B) HE-stained
tissue image, (C) 785nm laser + fluorescence image, (D) 405nm laser + fluorescence image, (E) 664nm laser + fluorescence image,(F) Fluorescence spectrum in the lymph node

─ 111 ─P-46
In vivo imaging of exosomes derived from stem cells by using quantum nano-optics
for regenerative medicine
Yuka Kumon,1 Hiroshi Yukawa,1,2,3 Onoshima Daisuke 1,2, Yoshinobu Baba1,2,3,4
Grad. Sch. of Engineering, Nagoya Univ., 2 Institutes of Innovation for Future Society, Nagoya Univ.
3
National Institutes for Quantum and Radiological Sci. and Tech. (QST), 4Health Research Institute, AIST1OBJECTIVE
In regenerative medicine, it is known that transplantation of stem cells into the whole body or the affected area
promotes regenerative therapeutic effect on the affected area by the grow factor produced by stem cells. In recent years,
it has been clarified that exosomes released from stem cells have a regenerative function, and administration treatment ofstem cells derived exosomes are attracting attention as a new therapeutic tool for regenerative medicine. However, theregeneration mechanism of stem cells derived exosomes remains unclear. The main cause is appears to be due to a lackof in vivo imaging and detecting technology of exosomes. In this study, we established a method of in vivo imaging of
exosomes with high sensitivity by using quantum dots (QDs). QDs have higher luminance than the conventional
fluorescent dyes and have wavelengths in the near infrared region (NIR). Furthermore, we established a more stablelabeling method by spontaneously incorporating them using glucose transporters expressed by exosomes.
METHODS
Glucose modification was performed on QD655 and the shape, particle size and zeta potential of Glucose-modified
QDs were measured using TEM and dynamic-light-scattering (DLS). For the introduction of glucose-modified QDs into
mouse adipose tissue-derived stem cells (mASCs), glucose-modified QDs containing medium were added into mASCs
and incubated at 37 °C for 3 hours. Then, the mASCs were washed by PBS in order to remove the remaining QDs, andQDs labeled mASC were evaluated by fluorescence observation, and flow cytometry. Exosomes were purified from
mASCs, and collected by ultracentrifugation. After suspending in PBS, exosomes were incubated with glucose-modified
QDs, at 37 °C for 3 hours and recollected by Exo-Quick for in vivo imaging of exosomes.
RESULTS
The particle diameter and zeta potential of QDs with and without glucose modification were 29.9 nm, 33.4 nm, and-29.5 mV, -19.0 mV, respectively. The diameter size of QDs were increased and zeta potential of QDs were decreased by
glucose modification. In addition, white contrast was confirmed around the QDs from the TEM image. These datasuggest that QDs were covered with glucose. Next, after the addition of glucose-modified QDs into mASCs, the red
fluorescence of QD655 was confirmed in mASCs by a confocal microscopy. In cytotoxicity test, the cell viability ofmASCs labeled with QDs remained almost 100% even at 24 nM of QDs. No significant differences in cell proliferation
rates between labeled and non-labeled mASCs was confirmed, therefore confirming that the glucose-modified QDs had
no effect the cell proliferation ability of mASCs. Moreover, the introduction efficiency of QDs to mASCs was 85.6% at
8 nM of QDs by a flowcytometry analysis. Similarly, the introduction efficiency of QDs to exosomes derived from
mASCs was showed 95.6% at the same concentration. On the other hand, the membrane of the QDs labeled exosome
was stained with a cell membrane staining reagent in order to perform double labeling, and observed with a superresolution microscope. The yellow fluorescence of exosomes labeled with glucose-modified QDs were observed by themerging of the red fluorescence of QD655 and the green fluorescence of cell membrane staining reagent (PKH67).
CONCLUSIONS
We succeeded in the establishment of a new introduction method of QDs into mASCs and mASC-derived exosomes
via glucose transporters. Glucose-modified QDs have no effect on cytotoxicity and self-proliferative ability, suggestingthat they are excellent quantum nano-imaging materials with high biocompatibility. The introduction-technology of QDs
inside exosomes by using glucose transporters is expected to be an extremely stable and highly sensitive imaging
technique in comparison to the conventional methods such as labeling of exosome membrane using fluorescence dyes.
─ 112 ─P-47
Ex vivo fluorescent imaging of macromolecule kinetics by nose-to-brain delivery withfunctional arginine-rich peptides
Takumi Kurano,1,2 Takanori Kanazawa,1,2,* Mami Kaneko,2 Hisako Ibaraki,2 Yuuki Takashima,2
Toyofumi Suzuki,1 Yasuo Seta,2
1Sch. Pharm., Nihon Univ., 2Tokyo Univ. Pharm. & Life Sci.
OBJECTIVE
Nose-to-brain delivery is a highly versatile route, which, in combination with novel macromolecule drugs being
developed for treating intractable CNS diseases, is a promising approach for the treatment of disorders. Furthermore,basic peptide carriers may improve nose-to-brain drug delivery by their capability to increase the stability of theencapsulated nucleic acids against chemical and biological degradation. In this study, to investigate the nose-to-brain
pathway using basic peptide carriers, the distribution kinetics of macromolecule in whole brain, nasal mucosa, andtrigeminal nerve after intranasal administration with arginine-rich peptide carriers modified with hydrophobic orhydrophilic moiety.
METHODS
We used CHHRRRRHHC peptide (CH2R4H2C) as arginine-rich peptide carriers, and modified with polyethylene
grycol-based block-copolymer (PEG-PCL) as hydrophilic moiety or stearic acid (STR) as a hydrophobic moiety. To
examine their utility as nose-to-brain drug delivery carrier, we compared the transmissibility of the model
macromolecules to the brain, we observed extracted each tissues following intranasal administration of model
fluorescein-macromolecule drugs (Alexa FluorⓇ647-dextran, Mw: 10,000) alone, or Alexa-dextran complexed with
PEG-PCL- CH2R4H2C or STR- CH2R4H2C to rats by fluorescent imaging system.
RESULTS
Significantly stronger fluorescence was observed in the whole brain, nasal mucosa and trigeminal nerve following
intranasal administration of Alexa-dextran with both peptide carriers than that of Alexa-dextran alone. In the rats
receiving the Alexa-dextran/PEG-PCL- CH2R4H2C complexes, strong fluorescence was observed in the not only nasal
mucosa and olfactory bulb but also trigeminal nerve. Furthermore, spreading of the fluorescence was observed after 30
min and 1 h in whole brain. Whereas, in the rats receiving the Alexa-dextran/STR- CH2R4H2C complex, although thefluorescence intensity was weaker in the trigeminal nerves, very strong fluorescence of Alexa-dextran was observed in
the nasal mucosa. In addition, strong fluorescence was observed in the olfactory bulb in the forebrain.
CONCLUSIONSBoth CH2R4H2C peptide-based carriers have also demonstrated the ability to improve the delivery of model drugs fromthe nose to the brain. In addition, PEG-PCL-CH2R4H2C can improve drug distribution throughout brain tissues after
movement from the nose to the brain compared with STR-CH2R4H2C. These results suggest that the enhancement ofdrug-delivery achieved via the nose-to-brain route may depend on the properties of the carriers.

─ 113 ─Grant & Foundation Acknowledgements
JSPS KAKENHI Grant Number 19HP0304

The NAGAI FOUNDATION TOKYOTERUMO LIFE SCIENCE FOUNDATION

Kato Memorial Bioscience Foundation

─ 114 ─Sponsor Acknowledgements
Sponsors with Exhibition and Advertisement
BizCom Japan, Inc.
CYBERDYNE, Inc.
Hamamatsu Photonics K.K.
HIGH-TECH CORPORATIONLightStone Corp.
NANOSEED
Nepa Gene Co., Ltd.
Summit Pharmaceutics International Corporation
Sysmex corporation
Yokogawa Electric Corporation

Sponsors with Advertisement
Bruker Japan K.K.
Olympus Corporation
Olympus Medical Science Sales Corporation
Ozakirika, Inc.
Thorlabs Japan Inc.
─ 115 ── 116 ─Author Index

A

Akazawa, Kazuki
Akimoto, Rena
Aoki, Kazuhiro
Aoshi, Taiki
Aubert, Isabelle

B

P-16
P-16
P-35
P-33
S1-4

G

Gotoh, Taku

Baba, Yoshinobu

P-38, P-46

Barzaghi, Paolo
Bhattacharya, Dipan
Brockman, Omer

P-3
PL1
P-40

C

Fujii, Tomoyuki
Fujishima, Hiroshi
Fujiwara, Hisashi
Fujiwara, Masazumi
Fukunga, Togo

H

Hagiwara, FumikoHagiwara, Yuki
Hamaguchi, Shogo
Hamano, Nobuhito
Hanamata, Shigeru
Hanaoka, Kenjiro
Haseda, Yasunari
Hasegawa, Akihiro

P-41
P-1
P-9
P-13
P-22, P-23

P-11, P-14

P-27, P-28, P-30,
P-31, P-39
P-21
P-19
P-32
P-22, P-23
S2-2
P-33
P-2

Chan, Hak-Kim
Chang, Young-Tae
Chen, Johnny
Chiba, Eri
Chiba, Yoko

P-42
S4-1
S6-3
P-28
P-7

Corti, Davide

S1-3

Hasegawa, Kota
Hashimoto, Kenji

S6-3
P-29
S6-3
S6-3
S3-3, P-15
S6-3

Hayashi, Yasuhiro
Hiiro, Haruhito
Hirai, Hirokazu
Hiruma, Shota
Homma, Koichi
Honda, Rena

P-20
S8-3, P-20, P-21,
P-22, P-23
P-29
P-19
P-1
P-24
S7-1
P-35

Horiguchi, Shuhei
Hosoya, Natsumi
Hossack, John
Hotta, Kohji
Humbel, Bruno

P-1
P-8
S6-3
P-37
P-3

D

Dasa, Siva
Deguchi, Yoshiharu
Diakova, Galina
Dixon, Adam
Doan, Thi Kim Dung
Du, Zhongmin

EEndo-Takahashi, Yoko P-32
Enoki, Ryosuke
S7-2
Enomoto, Hirofumi
S8-2
Ezure, Toru
P-10

F

I

Farry, Justin

S6-3

Ibaraki, Hisako
Iida-Tanaka, Naoko

Fibriansah, Guntur
Fujii, Hirofumi

S1-3
P-15

Iizumi, Mitsuru
Ino, Kosuke─ 119 ─P-47
P-17, P-18
P-37
P-44

Ishihara, Miya
Itay, Rachmilevitch
Iwano, Naoya

S4-3
P-40
P-34

Kowada, Toshiyuki
Koyama, Shohei
Kuchitsu, Kazuyuki

Kamada, Hajime

PL1
P-27, P-28, P-30,
P-31, P-39
P-40

Kudo, Nobuki
Kumagai, Ryoma
Kumamoto, Yasuaki
Kumon, Yuka
Kurano, Takumi

AL
P-33
S8-3, P-20, P-21,
P-22, P-23
S6-2
P-44
S3-1
P-46
P-47

Kamei, Yasuhiro
Kamikawa, Yuko
Kamimura, Masao
Kamiya, Mako
Kanamaru, Naohiro
Kanazawa, Takanori
Kanchanawong, Tony
Kaneko, Kiyoko
Kaneko, Mami
Kaneshiro, Junichi

P-12
P-12
S3-3, P-15
S1-2
P-26
P-47
S5-2
S1-1
P-47
P-1

Kurosawa, Toshiki
Kurusu, Takamitsu
Kusuhara, Hiroyuki

P-29
P-22, P-23
P-29

Kasai, Shigenobu
Kato, Koichi
Kato, Yasutaka
Katoh, Kaoru
Kawabata, Kenji
Kawakita, Ai
Kawakubo, Megumi
Kazunari, Furuya
Khan, Arshad

P-44
P-35
P-40
S5-3
P-29
P-26
P-17, P-18
P-45
S6-3

Kikkawa, Yamato
Kikuchi, Kazuya
Kinjo, Masataka
Kitamura, Kodai
Klibanov, Alexander
Kobayashi, Tomomi
Koizumi, Koji
Komoto, Shinya
Konishi, Yuki

P-32
P-16, P-43
P-4
P-38
S6-3
P-32
P-3
P-3
P-16

Masaki, Kojima
P-6
Matsudaira, Paul
PL1
Matsui, Akito
P-4
Matsumoto, Katsuhiko P-1
Matsumoto, Yoko
P-41
Matsumoto, Yu
S6-1
Matsumura, Yoshitaka P-6
Matsuo, Kazuya
P-24
Matsushita, Tetsuya
P-42

Konno, Ayumu
Kostyuchenko, Victor

P-1
S1-3

Matsuyama, Tetsuya
Meher, Akshaya

K

Kabla, Alexandre
Kageyama, Saori

L

Lim, Xin-Ni
Lim, Xin-Xiang
Liming, Li
Lok, Shee-Mei
Lu, Hoang D.

M

Mabuchi, Issei
Mafy, Noushaba
Mano, Tomoyuki
Maruyama, Kazuo

Maruyama, Tamotsu

─ 120 ─

S1-3
S1-3
P-45
S1-3
P-42

P-8
P-24
P-1
PL2, P-28, P-30,
P-31, P-32, P-39,
P-40, P-41
P-27, P-31

P-26
S6-3

Minoshima, Masafumi
Mitani, Tomoki
Mitsuyama, Totai
Miyakawa, Takuya
Miyamoto, Daisuke
Mizumaki, Toshiki
Mochizuki, Tatsuki
Mochizuki, Toshiaki
Morais, Marc

P-16, P-43
P-1
S5-3
P-7
P-21
P-38
P-29
P-3
S1-3

Moriyama, Kenji
Munakata, Lisa

P-40
P-27, P-28, P-30,
P-31, P-33, P-39
P-1
P-26

Murakami, Tatsuya
Murata, KaoriN

Okinaga, Hiroko
Okubo, Kyohei
Omata,Daiki

Ono, Seijiro
Onoshima, Daisuke
Onoue, Satomi
Oshimi, Keisuke

S1-1
S3-3, P-15
P-27, P-28, P-30,
P-31, P-33, P-39,
P-40, P-41
P-22, P-23
P-38, P-46
P-42
P-13

Osuga, Yutaka
Ozawa, Takeaki

P-41
S4-2

P

Prasad, Ankush
P-44
Prud'homme, Robert K. P-42

Nagai, Takeharu
Nakayama, Toshinori
Nakayama, Yoko

S4-4
P-2
S1-1

R

Namekata, Iyuki
Negishi, Yoichi
Ng, Thiam-Seng
Nigoghossian, Karina
Nishida, Haruka
Nishimura, Yushi
Nomizu, Motoyoshi
Nomura, Shintaro
Nonomura, Kenichi

P-19
P-32
S1-3
S3-3, P-15
P-41
P-13
P-32
P-11, P-14
P-23, P-22

S

Nuriya, Mutsuo

S7-3

Ristroph, Kurt D.

P-42

Sakamoto, Joe
Sato, Hideyuki
Sato, Teruya
Sawada, Jumpei
Sawada, Ryuji
Sekiya, Haruko
Seta, Yasuo
Seto, Yoshiki

P-12
P-42
P-44
P-22, P-23
P-10
S1-1
P-47
P-42

Shapira, YeruhamShi, Jian
Shibuya, Hirotoshi
Shigeto, Shinsuke
Shikoh, Eiji
Shiku, Hitoshi
Shima, Tadamitsu

Oda, Katsutoshi
Ogawa, Kazunori
Ogino, Hidetaka
Ohmori, Tamano
Ohtani, Naoko
Oka, Kotaro

P-41
P-22, P-23
P-2
P-19
S3-3
S5-4, P-36, P-37

Shimane, Yasuhiro

P-40
S1-3
P-11, P-14
P-5
P-13
P-44
P-27, P-28, P-30,
P-31, P-39
P-7

Okada, Yoshiaki
Okamoto, Koichi

P-33
P-26

Shindo, Hiroki
Shindo, Yutaka

P-20
S5-4, P-37

O

─ 121 ─

Soga, Kohei
Sone, Kenbun
Sugihara, Fuminori
Sugimoto, Kenji
Susaki, Etsuo A.
Suzuki, Mariko
Suzuki, Ryo

Suzuki, Shuji
Suzuki, Toyofumi
Suzuki, Yuno

T

S3-3, P-15
P-41
P-16
P-26
P-38
P-17
P-27, P-28, P-30,
P-31, P-32, P-33,
P-39, P-40, P-41

U

P-40
P-47
P-27, P-28, P-30,
P-31, P-39

Wada, Kenji
Watanabe, Kenshiro
Watanabe, Tomonobu
Wu, WeiUeda, Hiroki R.
Uehara, Ryota
Uehara, Tomoya
Ueki, Sota
Umezawa, Masakazu
Urano, Yasuteru

WY

P-1, P-38
P-24
S2-1
P-9
S3-3, P-15
S1-2

P-26
P-20
P-1
P-42

Takahashi, Sumika
Takamatsu, Tetsuro
Takashima, Yuuki
Takeshita, Norio

P-18
S3-1
P-47
P-5

Yagi, Hirokazu
Yagi-Utsumi, Maho
Yamada, Kohei
Yamada, Rino

P-35
P-35
P-42
P-35

Takuma, Saito
Tamaoki, Nobuyuki
Tamura, Masaru
Tan, Ter Yong
Tanabe, Ruriko
Tanaka, Hikaru
Tanaka, Masuaki
Tanaka, Minami
Tanaka, Yusuke

P-45
P-24
P-11, P-14
S1-3
P-11, P-14
P-19
P-13
S5-3
P-19

Yamada, Takumi
Yamaguchi, Kohei
Yamaguchi, Masayuki
Yamaguchi, Tomoko
Yamaguti, Mikiya
Yamamoto, Johtaro
Yamamoto, Shuhei
Yamanaka, Nobuaki
Yamanaka, Ryu

P-31
P-41
P-15
P-29
P-26
P-4
P-10
S3-2
S5-4, P-36, P-37

Tanikawa, Michihiro
Tanokura, Masaru
Tavakol, Sahar
Tega, Yuma
Teki, Yoshio
Tobita, Seiji
Toh, Bunki
Toyama, Yusuke
Toyota, Masatsugu

P-41
P-7
PL1
P-29
P-13
P-25
P-22, P-23
S5-1
S8-1

Yamashita, Atsushi
Yano, Yusuke
Yasuda, Mitsuru
Yasukagawa, Mami
Yeroslavsky, Gil
Yoshida, Kurumi
Yoshida, Michiharu
Yoshihara, Toshitada
Yoshioka, Yasuo

P-29
P-32
P-5
P-25
S3-3, P-15
P-43
P-40
P-25
P-33

Yukawa, Hiroshi

P-38, P-46

─ 122 ─Z

Zhang, Pingyu
Zhang, Shuijun
Zhong, Jun

S6-3
S1-3
PL1

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─ 133 ─2018年度決算書 (2018年1月1日～2018年12月31日）

日本バイオイメージング学会
会長

一般会計
収入

船津

高志

印

理事(財務担当） 太田

善浩

印

支出

2017年より繰越
利息
会費
学術集会余剰金
懇親会費

4,675,321
11
917,000
852,989
65,000

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ジャパンメディカル英文校正
通信・郵送費
謝金・人件費
男女共同参画141,10240,91983,252130,000
80,712

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6,510,321

奨励賞
会議費
振込手数料
学術集会準備金HP作成維持費
雑費

100,000
129,707
5,184
300,000
70,20056,757

特別会計(国際学会準備金等）
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2017年度より繰越

2019年度への繰越

5,372,488

支出計6,510,321

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2019年度への繰越

4,290,669

2019年
監事
監事

1,137,833

4,290,669

収入計

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小計

月
木原
高松

裕
哲郎

支出計

日
印
印
─ 134 ─4,290,669

4,290,669

2020年度予算案(2020年1月1日～2020年12月31日）
一般会計
収入
繰り越し
会費

収入計

支出
4,749,488
917,000

バイオイメージング印刷費
Bioimages アップロード費
ホームページ管理費
謝金・人件費
英文校閲費

150,000 広報
100,000 広報
70,200 広報
50,000 庶務、会計
80,000 編集

5,666,488

会議費
通信・郵送費
奨励賞･研究助成
男女共同参画（分担金 他）
学術集会準備金
雑費
予備費

130,000 庶務
110,000 庶務、会計
100,000 賞選考
99,000 男女共同・国際
300,000 集会
60,000 庶務・会計4,417,288

支出計

特別会計（国際学会準備金等）
収入
繰り越し
収入計

5,666,488

支出
3,290,669
3,290,669

2021年度への繰越
支出計─ 135 ─3,290,669
3,290,669

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バイオイメージング 第 28 巻第 2 号
2019 年 9 月 5
2 日発行
発行所：日本バイオイメージング学会〒223-8522 神奈川県横浜市港北区日吉 3-14-1
慶應義塾大学理工学部生命情報学科 生物物理・神経情報学研究室内
電話 ： 042-676-5498 ； FAX ： 042-676-5863
E-mail： office@j-bioimaging.org
URL： http://j-bioimaging.org/

こんな方にお勧めします 蛍光顕微鏡や蛍光実体でサンプルが暗いと感じている方 ディスク共焦点顕微鏡でもっと多くの色素を使いたいと思っている方 光刺激やオプトジェネティックをやりたい方

５つの特長
７波長（標準）、高輝度、安定、長寿命、低価格倒立型リサーチ顕微鏡

IX83/IX73
IX3ÊSeries

ライブイメージングの探求とともに進化する IX3
様々な研究のプロセスに対応し、拡張性豊かに研究者を支える
ライブイメージングのためのプラットフォーム、それが IX3シリーズです。
照明ムラや光ロスの少ない高品位で広視野なイメージング、
高精度かつ再現性のあるスピーディで快適なデジタルイメージングの実現など、柔軟なシステム拡張性で研究者のニーズと共に進化する確かな性能を提供します。

〒163-1412 東京都新宿区西新宿3-20-2 東京オペラシティタワー12F 機器事業本部 TEL : 03-3379-6003 FAX : 03-3379-6014

次世代型 共焦点レーザー走査型顕微鏡
FV3000シリーズ
新型分光システムTruSpectralと冷却GaAsP PMTによる圧倒的な明るさ
高感度・高精度のTruSpectral分光システム
2nmの高分解能と優れた透過率を実現する透過型回折格子を検出系に採用。
従来と比べ蛍光の検出効率が大幅に向上。

FV3000とFV1200の分光感度比較（FV1200を1とした比較）
1.5

集光レンズ

1.4
1.3
可変スリット: FV3000: FV1200

感度

1.2
1.1
1

VPH

0.9
400

450

500

550

波長 (nm)600

650

高感度冷却GaAsP PMT
光検出器の標準量子効率
40

量子効率 (%)

高い量子効率とペルチェ冷却によるノイズ低減、
高S/N実現するGaAsP PMT
を標準2Chで搭載。

GaAsP PMT

30
20
10
0
400

Multi-Alkali PMT
500

超高速イメージング * や2
広視野数18の高速イメージング * を実現するレゾナントスキャナー
1

600

700

波長 (nm)800

900

共焦点レーザー走査型顕微鏡 FV3000RS

マクロ 1.25倍からミクロ150倍、分解能約 120nmの超解像まで広い倍率レンジでのシームレスなイメージング

一般的なスキャナーの
視野数

対物

100倍に
切り換えて
ミクロ
樹状細胞（緑）とシナプス（赤）を可視化（共焦点画像）
観察
倍率そのままで超解像OSRイメージング
1.25倍拡大イメージ

FV3000
視野数 18, 8kHz
*1 最速438fps、512×32ピクセル *2 30fps、512×512ピクセル

マウス脳切片（1.25倍、ワンショットイメージ）〒163-0914 東京都新宿区西新宿2-3-1 新宿モノリス［お問い合わせ］お客様相談センター 0120-58-0414 受付時間 平日 8:45 ～17:30

超解像FV-OSRで細胞構造やシナプスをさらに高精細に可視化

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