Microbeam irradiation facilities for radiobiology in Japan and China.

Abstract

In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities have been developed in the world. This type of facilities now becomes an essential tool for studying bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to encourage collaborative research between radiobiologists interested in using microbeam in Japan and in China.

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http://naosite.lb.nagasaki-u.ac.jp/
NAOSITE: Nagasaki University's Academic Output SITENAOSITE: Nagasaki University's Academic Output SITE
Title
Microbeam Irradiation Facilities for Radiobiology in Japan and
China
Kobayashi, Yasuhiko; Funayama, Tomoo; Hamada, Nobuyuki;
Sakashita, Tetsuya; Konishi, Teruaki; Imaseki, Hitoshi; Yasuda,
Keisuke; Hatashita, Masanori; Takagi, Keiichi; Hatori, Satoshi;
Suzuki, Keiji; Yamauchi, Motohiro; Yamashita, Shunichi;
Tomita, Masanori; Maeda, Munetoshi; Kobayashi, Katsumi;
Usami, Noriko; Wu, Lijun
Author(s)
CitationJournal of Radiation Research, 50(Suppl.A), pp.A29-A47; 2009
Issue Date 2009-04
URL
http://hdl.handle.net/10069/22232
Description
Rights
Copyright (c) 2009 by THE JAPAN RADIATION RESEARCH SOCIETY
Versionpublisher

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J. Radiat. Res., Vol. 50, Suppl. (2009); http://jrr.jstage.jst.go.jp
J. Radiat. Res., 50: Suppl., A29-A47 (2009)
Microbeam Irradiation Facilities for Radiobiology in Japan and China
Yasuhiko KOBAYASHI1,2,3, Tomoo FUNAYAMA3, Nobuyuki HAMADA1,2,3,10,
Tetsuya SAKASHITA3, Teruaki KONISHI4, Hitoshi IMASEKI4,
Keisuke YASUDA5, Masanori HATASHITA5, Keiichi TAKAGI5,
Satoshi HATORI5, Keiji SUZUKI6, Motohiro YAMAUCHI6,
Shunichi YAMASHITA6, Masanori TOMITA7,
Munetoshi MAEDA7,8, Katsumi KOBAYASHI8*,
Noriko USAMI8 and Lijun WU9
Heavy-ion microbeam/Light-ion microbeam/X-ray microbeam/Bystander effects/Nucleus irradia-
tion/Cytoplasm irradiation.
In order to study the radiobiological effects of low dose radiation, microbeam irradiation facilities
have been developed in the world. This type of facilities now becomes an essential tool for studying
bystander effects and relating signaling phenomena in cells or tissues. This review introduces you available
microbeam facilities in Japan and in China, to promote radiobiology using microbeam probe and to enco-
urage collaborative research between radiobiologists interested in using microbeam in Japan and in China.
INTRODUCTION
Biological effect of low dose radiation has been attracted
much attention in modern human society, not only due to
high dependence to nuclear power as energy source, but also
due to realization of human activity in space. In order to
investigate mechanisms of biological effects, heterogeneity
of radiation dose in cellular level became a big barrier to be
overcome. This heterogeneity becomes apparent in relatively
higher dose in high LET, heavy-particle irradiation than in
low LET, electron or photon irradiation. From this rational,
microbeam irradiation facilities using proton or He ion were
developed in 1990s in Gray laboratory, UK1) and in
Columbia University, USA.2) Their pioneering works
revealed various interesting results concerning heteroge-
neous distribution of dose, or particle traversal per cell. Also
demonstrated in the reports are non-targeted effects, or
bystander effects, which was found as effects observed in
cells not directly hit or irradiated, but situated nearly the
cells actually received the radiation. As conventional radia-
tion biology is based on the premise that only those cell
nuclei exposed to radiation treatment are affected, the pres-
ence of bystander effects forces a paradigm shift and reeval-
uation of modern radiation biology.
So far, two types of micro irradiation facilities have been
developed widely in the world, one of which is high-LET
particle microbeam irradiation, and the other is soft X-ray or
X-ray microbeam irradiation. Several important evidences
have been discovered that have never been described in the
study using conventional irradiation. For example, the
studies have found that there is a mechanism to transport
bystander signals between the targeted cells and neighboring
untargeted cells through gap-junctional intercellular commu-
nication.3) It was also shown that a single hit cell among
thousands of cells could induce bystander effects, indicating
that there are soluble factors, secreted from the targeted
cells, which mediate non-targeted effects.4,5) Moreover,
nuclear micro-irradiation as well as cytoplasmic micro-
irradiation initiates bystander effects, implicating that not
*Corresponding author: Phone: +81-29-864-5655,
Fax: +81-29-864-2801,
E-mail: katsumi.kobayashi@kek.jp
1Department of Quantum Biology, Division of Bioregulatory Medicine,
Gumma Univ. Graduate School of Medicine, Maebashi, Gumma 371-8511,
Japan; 2The 21st Century Center of Excellence (COE) Program for
Biomedical Research Using Accelerator Technology;
Radiation Biology Group, Radiation-Applied Biology Division, Quantum
Beam Science Directorate, Japan Atomic Energy Agency (JAEA),
Takasaki, Gunma 370-1292, Japan;
Development, National Institute of Radiological Sciences, Chiba, Chiba
263-8555, Japan; 5The Wakasa Wan Energy Research Center, Tsuruga,
Fukui 914-0192, Japan; 6Atomic Bomb Disease Institute, Graduate School
of Biomedical Sciences, Nagasaki University, Nagasaki, Nagasaki 852-
8521, Japan; 7Cenral Research Institute of Electric Power Industry, Komae,
Tokyo 201-8511, Japan; 8Photon Factory, KEK, Tsukuba, Ibaraki 305-0801,
Japan; 9Key Laboratory of Ion Beam Bioengineering, Hefei, Anhui 230031,
China; 10Present address: Dept. Pathology, Institute of Development, Aging
and Cancer, Tohoku Univ. Sendai 980-8575, Japan.
doi:10.1269/jrr.09009S
3Microbeam
4Dept. Technical Support and

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only DNA damage but also dysfunction of mitochondria
plays a role in generating bystander signals.6,7) It is quite
clear that these findings have never been discovered without
targeted micro irradiation, and thus, microbeam irradiation
becomes indispensable for studying such non-targeted effects.
Soon after the construction of microbeam irradiation
facilities for raidiobiology in UK and USA, a project of
microbeam irradiation using heavy ions started in JAEA,
Takasaki in Japan. These facilities provided concrete
evidence on bystander effects and related phenomena, which
turned on the explosive progress in radiobiology in low dose
region. Since then, several proposals for constructing micro-
beam irradiation systems have been proposed in Japan, and
presently six systems are working and some more, under
development or being planned. One of the characteristics in
microbeam research status in Japan is that various types of
radiation are available, including soft X-rays, X-rays, light
ions and heavy ions. This indicates a potentiality of explo-
sive development in microbeam radiobiology in Japan. This
review introduces present status and characteristics of these
six microbeam facilities, all of which are open to outside
users under some sort of collaboration programs, in order to
encourage Japanese radiobiologists for using them and to
promote radiobiology in Japan. For the convenience of
readers, characteristics of these facilities are summarized in
one Table. Also added in this review is a microbeam facility
in Hefei, China, since China is not so far located for the
collaboration of Japanese scientists.
HEAVY-ION MICROBEAM SYSTEM AT JAEA-
TAKASAKI FOR BIOLOGICAL STUDIES
Introduction
Advantage associated with the use of heavy-ion micro-
beam irradiation concerns the precise detection of ion-hit
position on micron-scale targets. According to the model of
ion track structure, track structure of heavy ions is charac-
terized by a higher ionization density in the central part of
the track, called “core”, and a larger diameter of secondary
electrons called “penumbra” or “delta rays”, but the range of
energy deposition is no more than a few micrometers. On the
other hand, cell nuclei range from ten up to several tens of
micrometers in size. Therefore, when investigating bio-
logical effects together with the relationship to ion track
structure, it is important to know the precise position where
ion(s) hit. A precise regulation of the target position on the
cell nucleus by microbeam irradiation makes it possible to
obtain the information on the position of ion traversal and
on cellular responses induced by ion hit simultaneously,
indicating microbeam is an operative means for elucidating
initial cellular responses together with the relationship with
ion track structure.
Heavy ion-induced bystander effect is a phenomenon that
may be of concern to astronauts exposed to space radiation
during long-term space missions.8,9) On this context, the use
of heavy-ion microbeam is important for assessing health
risk of space radiation.
The use of heavy-ion microbeams has not been restricted
to the area of radiation biology and has been applied to other
areas including its use as a micro-radiosurgical tool to target
specific tissue regions of biological samples,10–15) as is the
case for heavy-ion radiotherapy. Heavy-ion microbeams can
inactivate specific cell populations in multicellular organ-
isms by targeted irradiation and allow for the investigation
of their function by observing changes in the irradiated tar-
gets. Similar analyses can be performed using micro laser
ablation techniques. However, micro laser irradiation com-
pletely eliminates target cell and tissue structures as a result
of heat-induced degeneration, while we can suppress cell
division and gene expression without destroying intact tissue
structures and intercellular interaction by heavy-ion micro-
beam irradiation. Moreover, the heavy-ion energy deposited
at the Bragg peak can be controlled by adjusting the ion
energy. Thus, heavy-ion microbeam irradiation can be
employed in precisely controlled microsurgical operations
with fewer side effects compared with micro laser ablation
techniques.
Heavy-ion microbeam cell targeting system at JAEA-
Takasaki
The heavy-ion microbeam system at JAEA-Takasaki
employs a micrometer-sized collimator (microaperture) for
List of microbeam facilities for radiobiology in Japan and China
Institute or UniversityLocation Type of Beam and Its Energy MinimunBeamSize
TIARA, Japan Atomic Energy AgencyTakasaki, JapanC (18.3 MeV/u), Ne(17.5 MeV/u), Ar(11.5 MeV/u) 5 μm
SPICE, National Institue of Radiological ScienceChiba, Japanproton, 3.4 MeV 5 μm
Wakasa Wan Energy Research CenterTsuruga, Japan proton(10 MeV), He (15MeV) 10 μm
Nagasaki UniversityNagasaki, Japan ultrasoft X-rays, 0.25 keVa few μm
Central Research Institute of Elec. Power Ind.Tokyo, Japan soft X-rays, 1.49 keV 2 μm
Photon Factory, KEK Tsukuba, Japan X-rays, 4 - 20 keV5 μm
Key Lab. Ion Beam Bioengineering(LIBB) Hefei, Chinaproton, 2-3 MeV5 μm

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generating heavy-ion microbeams from ions accelerated
using the azimuthally-varying-field (AVF) cyclotron.16–18)
This system has been used by JAEA-Takasaki group to irra-
diate a variety of samples including cultured cells,16,19–21)
nematode,10) plant tissue11,15) and silkworm.12–14) The appara-
tus can generate microbeams with most of the ion species
accelerated by the AVF cyclotron using various microaper-
ture sizes ranging from 5 to 250 μm in diameter. The ion
species frequently used include carbon (18.3 MeV/u), neon
(17.5 MeV/u) and argon (11.5 MeV/u). For the irradiation of
biological samples, ions are extracted into air from vacuum
via a 8 μm-thick polyimide window or, in case of beam sizes
less than 20 μm, extracted directly into air by maintaining
the vacuum of the beam line through differential pumping.
Irradiated ions penetrate cell samples cultured on the plastic
ion track detector CR-39 (TNF-1, 100 μm thick), and are
then detected and counted by the PMT-scintillator assembly
installed on the revolver of the optical microscope used for
cell irradiation. The system is designed for the rapid and
simultaneous detection of ion hits on cell samples by visu-
alizing the track on the counter side of CR-39 base plate as
etched pits using an alkaline-ethanol solution under cell cul-
ture conditions (37°C, 5% CO2 and 100% humidity). The
variable beam size implemented by the exchangeable
microaperture enables the system for use with a variety of
biological targets from cultured cells in the micrometer-scale
to silkworm larva in the millimeter-scale. Cells are stained
with the fluorescent vital staining dye CellTracker Orange to
locate the targets, and the position of the cells is then deter-
mined by image processing to generate the coordinate data-
base using the “offline” microscope system, which is located
away from the beam line to facilitate rapid irradiation.
Following this, samples are transferred onto the “online”
microscope, and then rapidly and automatically irradiated
according to the coordinates stored in the database generated
at the “offline” system. Because the position of beam is
fixed, irradiation of sample is carried out by systematically
moving each sample by the sample stage to the position of
the beam. Target samples have included not only the cul-
tured cells for radiobiological study, but also the root tissues
of the thale-cress Arabidopsis thaliana, embryos and larvae
of the silkworm Bombyx mori, and the nematode
Caenorhabditis elegans, to investigate biological phenome-
na pertaining to a diverse group of fields including develop-
mental biology, neurobiology and plant physiology. Detailed
experimental procedures are described previously.18)
Another microbeam system we are working on is newly
developed focused heavy-ion microbeam system.22,23) The
system is equipped with a magnetic quadrupole quadruplet
lens system for higher spatial resolution and with an X, Y
beam scanner for fast hitting of single ion to micron scaled
samples like a biological cell. In vacuum, a microbeam gen-
erated by the system had spatial resolution of less than 1 μm.
To irradiate this finer microbeam to the specific region of
individual cells, a new cell targeting system was designed
and installed under the beam extraction window. The system
consists of Olympus IX81 full-automatic inverted micro-
scope system, and the set of automatic stages for managing
sample and microscope alignment. The system is settled on
the mount frame that is rigidly fixed to the quadrupole qua-
druplet lens for avoiding influences of vibration.
Biological applications of heavy-ion microbeam
Studies of direct hit effects of heavy ions
Chinese hamster ovary (CHO) cells were targeted with
40Ar ion microbeams of 5 μm in diameter (11.5 MeV/u,
LET = 1260 keV/μm), and the precise position of ion hits
was determined by merged images of the cells and ion-pits
etched on a CR-39 plastic ion track detector, showing that
nuclear hits, comprising a single ion, and cytoplasmic hits
significantly suppressed cell growth.16) In addition to mam-
malian cultured cell lines, other cell types have been irradi-
ated under in vitro culture conditions. As a model system
representing single plant cells, tobacco BY-2 protoplasts
were targeted with 12C ion microbeams 20 μm in diameter
(18.3 MeV/u, LET = 121 keV/μm) to investigate the clono-
genicity of targeted cells.20) As a model system for the inves-
tigation of muscular dystrophy where microinjury of the
plasma membrane occurs,24) single fibers isolated under in
vitro culture conditions from mouse skeletal muscle were
irradiated with 20Ne (12.8 MeV/u, LET = 375 keV/μm) and
40Ar (11.5 MeV/u, LET = 1260 keV/μm) ion microbeams 20
μm in diameter, and ultrastructural changes were examined
using electron microscopy. We observed irregular protru-
sions and invaginations in the plasma membrane, irregular
disruption of microfilaments in the cytoplasm near the
plasma membrane, and multiple autophagic vacuoles. These
findings suggest that heavy-ion irradiation causes disruption
of the cellular architecture, and the removal of which
involves autophagy.24)
Studies of the bystander effects
With less than 0.02% of confluent AG01522 fibroblasts
targeted with 20Ne (12.8 MeV/u, LET = 375 keV/μm) and
40Ar (11.5 MeV/u, 1260 keV/μm) ion microbeams 5 μm in
diameter, we found that gap junctional intercellular com-
munication and reactive oxygen species mediate bystander-
induced micronucleus formation.19,21) Recently, with less
than 0.01% of confluent AG01522 fibroblasts targeted with
12C (18.3 MeV/u, LET = 103 keV/μm), 20Ne (17.5 MeV/u,
294 keV/μm) or 20Ne (13.0 MeV/u, 375 keV/μm) ion micro-
beams 20 μm in diameter, we showed that bystander effects
manifested itself as inactivated clonogenic potential, a
transient apoptotic response and delayed p53 phosphoryla-
tion,25,26) and that gene expression profiles in bystander cells
are substantially different from those in irradiated cells.27)
We have also shown bystander-induced suppression of cell
proliferation in CHO cell cultures using 40Ar ion micro-
beams 5 μm in diameter (11.5 MeV/u, LET = 1260 keV/

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μm).16) With the in vivo targeting of germline cells in the
nematode using 12C ion microbeams 20 μm in diameter
(LET = 120 keV/μm), we observed little, if any, bystander
effects in nonirradiated bystander tissues.10)
Studies using radio-microsurgical techniques
An example of plant physiological research by our group
concerned the investigation of root gravitropism in thale
cress.11) Primary root apical tissues targeted with 12C ion
microbeams 120 μm in diameter (18.3 MeV/u, LET = 110
keV/μm) significantly suppressed root elongation and curva-
ture at the root tip. Irradiation of cells that would later form
the lower part of the root cap following gravistimulation
resulted in dramatic inhibition of root curvature, an effect
not observed following irradiation of cells that would form
the upper part of the root cap. Targeted exposure to narrower
microbeams (40 μm in diameter) revealed that inhibition of
curvature was most pronounced at the root tip, followed by
cells in the lower part of the root cap. These findings suggest
that the most sensitive sites related to root gravity comprise
the root tip and columella cells, and that the root gravity
signaling pathway traverses the lower part of the cap cells
following perception.11) Recently, we also analyzed the
function of the root cap and elongation zone cells in root
hydrotropism using our 180-μm-diameter microbeams of 12C
ions (LET = 135 keV/μm).15) Targeted irradiation of the elon-
gation zone, but not the columella cells, significantly inhibit-
ed the development of hydrotropic curvature. Laser ablation
as another microsurgical approach revealed that columella
cells are indispensable for hydrotropism. Thus we showed
that both the root cap elongation zone play indispensable and
functionally distinct roles in root hydrotropism.15)
To apply microbeam technology in the area of insect
developmental biology, we investigated embryogenesis in
the silkworm.12) To this end, various sites within the eggs
were exposed to 12C ion microbeams (LET = 110–200 keV/
μm) collimated using microapertures of varying diameter
ranging from 60 to 250 μm. Targeted irradiation resulted in
the generation of abnormal embryos which exhibited local-
ized defects of organs including deletion, duplication and
fusion in a manner dependent on the dose, beam size and
choice of target site. Taking into account the close correla-
tion between the location and frequency of these phenotypic
defects on the resulting embryos and the targeted sites, we
succeeded in establishing a fate map for the cellular blasto-
dermal stage embryo.12) The knob-forming region in first
instar larvae of the knob mutant silkworm, which exhibit
knobs on the dorsal side of larva spots, were also targeted
with 12C microbeams 180 μm in diameter (LET = 128 keV/
μm), and knob formation was found to be suppressed at the
irradiated segments.14)
To evaluate the radiation effect on individual organisms,
we investigated positional radiation effects on nematode ger-
mline cells.10) In this study, germline cells in the gonad were
irradiated with 12C ion microbeams 20 μm in diameter (LET =
120 keV/μm). Targeted irradiation of the tip region of the
gonad arm at the L4 larval stage arrested germ cell prolifer-
ation, while irradiation of the pachytene region at the young
gravid stage induced apoptotic cell death in the gonad. This
was also observed in the c-abl-1 mutant nematode.10) Thus,
radio-microsurgical approaches employing targeted irradia-
tion with heavy-ion microbeams generated at JAEA-Takasaki
proved to be useful in characterizing the tissue-specific,
local biological response in eukaryotes.
SINGLE PARTICLE IRRADIATION SYSTEM
TO CELLS: SPICE, AT NIRS
Outline of the single particle irradiation system to
cells: SPICE
An electrostatic accelerator facility of NIRS supplies pro-
tons and helium ion beam with a Tandetron accelerator
(High Voltage Engineering Europa B.V.) at maximum ener-
gies of 3.4 MeV and 5.1 MeV, respectively. In this PIXE
Analysis system and Tandem Accelerator (PASTA) facility,
there are four beam lines,28) and its three horizontal beam
lines, conventional (in vacuo), in air and droplet29), and
microbeam scanning beam line30) are available for PIXE
analysis. In the fourth beam line, a single particle irradiation
system to cells, SPICE has been constructed.31,32) The beam
is transported upward with a 90 degrees bending magnet
installed in the middle of the microbeam scanning PIXE
beam line. In order to get a microbeam in SPICE, the beam
is focused by a mono bloc triplet lens so as to exclude low-
energy, scattered particle components as often seen in other
microbeam facilities utilizing collimation methods.
Beam size measurements using CR-39
The beam size was determined by irradiating a plastic
track detector, CR-39. A CR-39 (100 μm thick) was adhered
to the cell dish instead of cells, and then the dish was set on
the voice coil motor-driven sample stage. The gap between
the beam exit window, Si3N4 membrane (1 μm thick), and
the targeted CR-39 was approximately 300 μm. Then, CR-
39 was etched in 7M NaOH at 70°C for 2 hours and its
image was obtained by confocal laser microscope (FV1000,
Olympus). Figure 1 shows an image of irradiated CR-39
after the etching procedure. For each position, protons were
irradiated by moving the sample stage with 50 μm pitch.
Irradiation was performed automatically according to a text
file containing data on a preset number of protons and on the
X-Y coordinates of the sample stage position. This system
enables one to irradiate 6–8 positions per second.
Cell targeting and irradiation system
For the routine irradiation on cells, the beam intensity is
controlled to be below 5.0 × 104 protons per second. The
number of protons having traveled through the cells is count-
ed using a scintillation detector equipped on a microscope

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system, which was set above the cell dish. Configuration of
the components around the beam exit, such as the micro-
scope, voice coil motor-driven sample stage, scintillation
detector, and cell dish, are shown in Fig. 2. The system con-
trolling PC controls a voice coil motor-driven sample stage,
which was developed with Techno-hands, Co., Ltd., and also
controls a fast beam deflector (10 MHz), which was installed
upstream of the 90-degree bending magnet, with high-speed
trigger pulse to turn on/off the beam. It is possible to irradi-
ate with precise number of protons from one to an arbitrary
number per position or cell. The nuclei of the cells are dyed
with Hoechst 33258, and fluorescent images were captured
by a 20× objective lens and a CCD camera (ORCA-ER,
Hamamatsu) equipped on the microscope (BX51, Olympus).
The size of single image frame was 430 μm × 330 μm, and
the fluorescence of dyed cell nuclei within 3 mm × 3 mm
area can be captured by taking 7 × 9 images. From these
images, the X-Y coordinates of the cell position according
to the fluorescence are calculated. An example of fluores-
cence image of nuclei of CHO-K1 cells dyed with Hoechst
33258 is shown in Fig. 3. The surrounding ellipse of each
cell nucleus was drawn using self-developed cell recognition
algorism based on a least-squares technique.33) Each nucleus
was numbered to identify the cells for irradiation experi-
ments afterward. All of the irradiation procedures can be
performed automatically after setting some parameters, such
as a preset number of protons.
Cell dish and sample preparation
The cell dish was designed to culture a larger number of
cells in a single dish and to reduce the air gap between the
cells attached on the bottom of dish and the beam exit win-
dow. The air gap is adjusted to be below 300 micrometers
to reduce beam scattering by the air gap. Photograph of cell
dish is shown in Fig. 2B. Dish for the system was designed
so as to sandwiching 2.5 μm thick Mylar film between a 30
mm diameter steel ring and a 33 mm diameter hole. Cells
can be cultured on a area of 30 mm in diameter. On the sur-
face opposite to the cell, blue lines are drawn with a perma-
nent marker to define the origin of coordinates in the cell
dish. This origin is used for describing the X-Y coordinates
of the cells in the dish in imaging process for cell targeting
and cell observation. Cells are seeded in this dish at 37°C
under 5% CO2 for 5 hr before irradiation, and stained with
1 μM Hoechst 33258 1 to 2 hrs prior to irradiation. Just
before irradiation, the media were replaced with 1 ml of
phosphate buffered saline (PBS). After 6 μm thick polypro-
pylene (PP) film was floated on the surface of the PBS, PBS
was removed, so that the cells were covered with PP film to
avoid them from drying. Cell dish thus prepared is placed on
the sample stage for irradiation.
Preliminary cell irradiation experiment
In order to confirm the performance of the system, reac-
tive oxygen species, ROS, produced in the cell irradiated
with microbeam were detected. ROS are important for
understanding the effects of radiation-induced cellular
damage. Many fluorescent probes have been developed for
this purpose.34) Among them, 2,7-dichlorofluorescin dia-
cetate (DCFH-DA), has been used frequently in radiation
biology for quantifying oxidative events in cells. DCFH-DA
is absorbed by cells, and is rapidly deacetylated by intracel-
lular esterases to 2,7-dichlorofluorescin (DCFH); subse-
quent oxidation of DCFH produces the fluorescent product
2,7-dichlorofluorescein (DCF).35) We applied this method to
Fig. 1. Image of irradiated CR-39 after the etching procedure. 10
different positions on CR-39 were aimed at by moving the sample
stage with 50 μm pitch. Number of protons irradiated for each posi-
tion are indicated in the figure. Bar size, 50 μm.
Fig. 2. Panel A is a drawing of the setup around the beam exit. Panel B is a photograph of the cell dish.

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detect ROS induced intracellularly with 3.4 MeV proton
microbeam.
CHO-K1 cells were cultured in α-MEM containing 10%
fetal bovine serum with appropriate antibodies. To detect the
3.4 MeV proton-induced of ROS with DCFH-DA probes,
cells were seeded in a microbeam dish 24 hr prior to irradi-
ation. Approximately 80% of cells were confluent at the
time of irradiation. Approximately 2 hrs prior to irradiation,
the medium was changed to medium containing 1 μM Hoe-
chst 33258, and 30 minutes prior to irradiation, the medium
was replaced with DCFH-DA solution (100 μM DCFH-DA
in PBS). The targeting procedures were completed in less
than 5 minutes after placing the cell dish onto the sample
stage. Targeted cells were selected manually on the fluores-
cence image of dyed cells. Figure 4A shows the fluorescent
image obtained by the microscope, and a) – d) are the four
cell nuclei targeted. The preset numbers of protons for the
targeted cell nuclei, a)–d) were 5 × 104, 1 × 105, 2 × 105,
5 × 105, respectively. Figure 4B shows the fluorescent prod-
uct DCF, which was the result of ROS production by the pro-
ton traversal through the cell nuclei. The intensity of fluo-
rescence in those irradiated cells was shown to increase with
increasing number of protons irradiated. Further investiga-
tion is under way concerning the detection of induced DNA
damage, cell cycle arrests, and cell inactivation with irradi-
ation of below one hundred protons.
Summary and future work
We have developed the microbeam irradiation system,
SPICE. Approximately 5 μm beam in diameter is available
now, and irradiation with single proton can be performed.
The maximum speed for cell irradiation is 400–500 cells per
minute with aid of automated imaging sysytem, and most of
the irradiation procedures can be performed automatically
by setting some parameters. As a demonstration, CHO-K1
cells were irradiated and the production of reactive oxygen
species ROS in the targeted cells was detected. Further
improvements are on the way, such as an off-line microscopic
system for post-irradiation biological analysis, improve-
ments on targeting accuracy and its beam size below 2 μm
in diameter. SPICE and PASTA are now an open facility for
collaborative researches.
SINGLE CELL IRRADIATION SYSTEM AT THE
WAKASA WAN ENERGY RESEARCH CENTER
Introduction
Recently, the application of microirradiation techniques in
biology has attracted much interest, as such techniques make
it possible to irradiate individual cells with ions under pre-
cise dose control, thus providing a unique opportunity to
study the inter- and intracellular responses of individual cells
to low-dose irradiation. A number of microirradiation sys-
tems for radiobiology have been developed and are now
Fig. 3. Example of the fluorescent image of the cell nuclei
obtained with the microscope system. The surrounding ellipse of
each cell nucleus represents the results of automated cell recogni-
tion, and the identification numbers and X-Y coordinate are com-
puted automatically.
Fig. 4. Panel A shows the fluorescent nuclei image Targeted nuclei are indicated as a) – d) in Panel A,
which were irradiated with 5 × 104, 1 × 105, 2 × 105, 5 × 105 protons, respectively. Panel B shows the fluo-
rescent product, DCF, which was the result of ROS production by proton traversal through the cell nuclei.

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operational.1,2,36,37) A single-cell irradiation system is cur-
rently under development at the Wakasa-wan Multi-purpose
Accelerator with Synchrotron and Tandem (W-MAST).38)
Cells are irradiated using a vertical ion beam, with collima-
tion achieved using a glass capillary.39) This report presents
the design and construction of this single-cell irradiation
system, and provides some results demonstrating the capa-
bilities of the system.
Experimental facility
Beam line
The beam line for cell irradiation is installed in Irradiation
Room 2 at the W-MAST facility. A proton or helium beam
accelerated by the tandem accelerator is used for single-cell
irradiation. The linear energy transfer (LET) in the water and
range are 4.7 keV/μm and 1.2 mm for the 10 MeV proton,
and 41 keV/μm and 215 μm for the 15 MeV helium ion,
respectively. Figure 5 shows a side view of the beam line.
The beam, transported vertically, is collimated with two
apertures of 2 mm and 0.5 mm diameters. The distance
between two apertures is 346.5 mm. After rough collima-
tion, beams are extracted in air through a glass capillary with
a 5 or 10 μm inner diameter. The angular spread of the ion
beam due to the geometry of the collimators is estimated to
be 1 mrad. Tilt and position of the glass capillary are adjust-
able, and the glass capillary is aligned so that the energy
spread of the collimated beam is minimized. After collima-
tion, the beam is extracted into air through a Mylar foil and
irradiated to the cell.
Stage and microscope
The stage for cell irradiation is installed immediately
below the beam exit. The stage is driven by motors and can
move in the x, y, and z directions. A size of the sample stage
is 246 mm × 246 mm, and dishes of 60φ or 35φ, or chamber
slides can be settled on the stage. Cells are cultured on a
CR39 sheet, which is pasted on the bottom of a perforated
dish. When the cells are irradiated, they are covered with a
polyimide foil for the prevention of dehydration. An optical
microscope with a charge coupled device (CCD) camera is
mounted beneath the irradiation stage to obtain images of
living cells. A silicon surface detector (SSD) is also mounted
in one of the ports on the objective-lens revolver of the
microscope. The SSD is covered with an Al foil of 2 μm
thickness for the light shield. The SSD is utilized for the
measurement of the energy spectrum of the collimated beam
and for the ion counting. Images of cells are processed and
viewed on a PC, and the positions of individual cells are cal-
culated in stage coordinates. The determination of the beam
position is performed by observing beam fluorescence of a
plastic scintillator placed at the target position. Before the
irradiation, we observe cells with the microscope, move
them to the beam position and record coordinates on the
stage. At the irradiation, the number of incident ions is
counted with the SSD.
Single event control
The number of ions incident at the cell is controlled by
electrostatic deflection of the ions together with ion detec-
tion using a silicon surface-barrier detector (SSD). Figure 6
shows a schematic diagram of the control system. The elec-
trostatic deflector, which is used for the beam blanking, is
installed on the injection line into the accelerator. When the
cell is irradiated, the number of ions which have penetrated
through the cell is counted using the SSD. When the number
of signals reaches a preset number, the output signal from
the preset scaler activates the high-voltage unit driving the
electrostatic deflector. For single-event control, it is neces-
sary to irradiate cells at a rate of less than 100 ions/s due to
the relatively slow response (~1 ms) of the high-voltage unit.
Sequential irradiation control
Individual cells at different positions on the stage can be
irradiated sequentially using the automatic control system
shown schematically in Fig. 6. Automatic control is per-
formed via a PC equipped with 4 peripheral boards provid-
ing digital I/O (DIO), counter, serial interface and video
capture functions. The motor controllers for the stage move-
ment and the image-processing unit for the optical micro-
Fig. 5. Side view of the single cell irradiation beam line. B, Q, S
denote bending, quadruple and steering magnets.
Fig. 6. Schematic diagram of the single-cell irradiation system.

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scope are connected to the PC via an RS-232C interface. The
DIO board is connected to a NIM module that generates a
control signal for the high-voltage unit driving the electro-
static deflector. Before irradiation, the position data for indi-
vidual cells are input into the automatic control software.
Irradiation is started after positioning of the stage. When the
number of ions detected with the SSD reaches a preset
number, the output signal from the preset scaler is received
by the DIO board to activate the high-voltage module for the
electric deflector. Then the motor controller moves the stage
under automatic control to the next irradiation position.
After the movement is complete, the DIO board outputs a
signal to deactivate the electrostatic deflector and thereby
start irradiation on the next cell. The number of protons for
each cell position is recorded in the PC.
Results of experimental tests
At first, energy spectrum of the collimated beam was mea-
sured using the SSD. The helium beam with energy of 15
MeV was extracted into air through a Mylar foil of 2 μm in
thickness. The glass capillary of 5 μm inner diameter was
used for the measurement. The SSD was positioned in air
15 mm below the exit window. The measured energy spec-
trum shows that 93% of the detected helium ions are dis-
tributed near the high-energy end, forming a peak in the
spectrum. In the case of the proton beam with energy of 10
MeV, 78% of the detected protons are in the peak at the
high-energy end using the glass capillary of 10 μm inner
diameter.
The beam sizes were measured using CR-39 track detec-
tors placed about 1 mm and 2 mm below the beam exit
window when 15-MeV helium beam was extracted through
the capillary of 5 μm inner diameter. After etched in 6 N
NaOH at 60°C, the track distribution observed under a laser
microscope (Fig. 7) shows that the beam spread was about
10 μm on the CR-39 placed 1 mm below the beam exit
window. When placed 2 mm below of the beam exit, the
beam spread was about 20 μm. The beam sizes thus esti-
mated were larger than the inner diameter of the capillary (5
μm). The angular spread of protons due to multiple scatter-
ing in the beam path, especially at the beam exit window, is
considered to be responsible for this beam broadening.
Figure 8 shows the incidence pattern obtained by auto-
matic irradiation of the CR-39 track detector to draw the let-
ters ‘WERC’. The letters were about 100 μm tall each, and
consisted of total 76 points, with 50 counts per point. The
letters ‘WERC’ can be recognized clearly, which shows that
the automatic irradiation is performed successfully. Tracks
distributed randomly around the letters are presumably due
to edge scattering at the capillary.
Summary
A system for irradiating single cells with controlled ion
doses has been developed at the W-MAST facility. The col-
limated ion beam delivered vertically is used for the irradi-
ation. The system is controlled using a PC and NIM modules
to allow sequential cell irradiation. The results of test exper-
iments revealed that 93% of the collimated helium ions were
mono-energetic and that the beam spot was about 10 μm.
Automated irradiation of a CR-39 track detector demon-
strated the sequential irradiation successfully. Further deve-
lopments for the determination of the beam position at the
target position and for observing living cells with the micro-
scope are needed for the single cell irradiation.
NAGASAKI UNIVERSITY SOFT X-RAY
MICROPROBE
Composition of the system
There are a couple of microbeam facilities in the world
including those settled in Nagasaki University. Nagasaki
University had conducted so called the 21st century Center
Of Excellence (COE) program between 2002 and 2007. The
title of the program was “International Consortium for Med-
ical Care of Hibakusya and Radiation Life Science”, and, in
Fig. 7. Beam size measured using a CR-39 track detector. Bar is a
10 μm-long scale.
Fig. 8. Incidence pattern after automated irradiation of the letters
‘WERC’. Each letter is about 100 μm tall.

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order to promote researches toward low dose radiation
effects, Nagasaki University COE program introduced Soft
X-ray microprobe, which was originally developed in Gray
Cancer Institute in United Kingdom.40) The Nagasaki Uni-
versity soft X-ray microprobe consists of three major parts,
which are the microfocus X-ray source, the X-ray focusing
assembly, and the cell imaging and alignment unit. Focused
electron bombardment of a carbon target generates charac-
teristic X-rays (278 eV). A continuum of bremsstrahlung is
removed by reflecting the radiation off with a 25-mm-
diameter silica mirror placed between the target and the X-
ray focusing assembly. Focused X-rays are obtained using
zone plate, which is made from tungsten deposited on a 100-
nm-thick silicon nitride substrate, together with the order
selecting aperture (OSA). Cells are cultured on a 0.9-μm-
thick mylar membrane, firmly placed at the bottom of metal
dish, which is clamped to a two-axis, stepped-motorized
microscope stage. They are stained with the DNA-binding
dye Hoechst 33258, and the nuclear image is captured by an
intensified charge-coupled device. Then, the experimenter
selects a cell or cells to be targeted using an on-screen com-
puter mouse pointer. As reported previously, dose distribu-
tion to the cell is not homogeneous. For example, about 40%
of the dose is deposited in the first micrometer of the cell.
About 50% of the dose is absorbed in the lower half of the
nucleus, while 6% is absorbed in the upper half. Based upon
the absorbed energy in the nucleus divided by the nuclear
mass, a Chinese hamster V79 cell would receive 1 Gy with
about 10,000 photons.
Localized Photon Delivery System (LPDS)
Recent studies using soft X-ray and alpha-particle micro-
beam irradiation further extended our knowledge of radia-
tion effects.41,42) However, the application of micro-irradiation
technique is not only limited to the studies in the field of
radiation biology. As microbeam is capable of depositing
radiation energy to a very limited area within a cell, micro
irradiation, we now call this system as localized photon
delivery system (LPDS), provides unique opportunity to
investigate physiological functions of micro-components of
the cell constituents. For example, while DNA has been
treated as a primary target for radiation exposure, there are
increasing numbers of literature reporting a role of protein
damage on radiation effects.43–45) It is likely that ionized pro-
teins reveal altered functions. In fact, long-lived protein
radicals are supposed to be involved in radiation-induced
mutagenesis.43) Because LPDS deposits radiation energy
only to the nucleoplasm or cytoplasm, it can separate radia-
tion effects stem from DNA damage and protein damage. It
was also shown that extremely low-dose radiation stimulates
growth factor receptors, which result in activation of
mitogen-activated protein kinase pathway.44) LPDS is able to
deposit dose only to the membranes. Thus, LPDS is highly
expected to be applied to the experiments investigating such
non-DNA effects. Moreover, advanced technology using flu-
orescence protein-tagged proteins enables visualization of
target proteins in a living cell. For example, an expression
of EGFP-tagged tubulin illustrates spindle fibers, whose dys-
function causes mitotic defects. Microirradiation to a part of
spindle fibers may lead to miss-segregation of the chromo-
somes. Furthermore, as a pioneer study lead by Cremer has
proven its possibility,46) LPDS could be a clue to depict
higher-order chromatin organization in the cell nucleus.
Since the width of the finest microbeam is expected to be
sub-micrometer, which corresponds to the size of several
megabase-chromatin domains, it is possible to examine the
effects of disorganization of chromatin domains on a sta-
bility of chromatin territory, chromatin movement, and on
interaction of damaged chromatins. Although previous
studies have applied UV laser for such purpose, future stud-
ies of chromatin dynamics cannot be accomplished without
PLDS.
Biological results and future perspectives
Our group has been applied LPDS for investigating DNA
damage response in mammalian cells. As reported prev-
iously, radiation-induced DNA double strand breaks initiate
activation of DNA damage checkpoint pathway. DNA
damage-induced higher-order chromatin disorganization dis-
sociates ATM dimers or oligomers to the monomers, whose
process is essential for the execution of DNA damage
response. Activated ATM through autophosphorylation at
serine 1981 phosphorylates downstream effectors, such as
histone H2AX, 53BP1, MDC1, NBS1, and p53. It has been
shown that these phosphorylated effectors as well as phos-
phorylated ATM form discrete foci, which are detectable as
dotted signals by immunofluorescence analysis. Foci of each
factor are always co-localized, indicating that foci formation
is phosphorylated ATM-dependent. Because the number of
foci is equivalent to that of DNA double strand breaks, it is
generally believed that the foci visualize chromatin regions
that have DNA double strand breaks. We have reported the
dose-dependent induction of phosphorylated ATM foci, and
more recently, we successfully visualize phosphorylated
ATM- and phosphorylated histone H2AX-dependent forma-
tion of 53BP1 foci in situ, by introducing the EGFP-tagged
53BP1 gene into normal human diploid cells. Our current
study illustrates dynamic process of 53BP1 foci formation,
whose dynamics is tightly related to the cell cycle check-
point regulation. Such studies using live cell imaging tech-
nology must be the future trend to understand the dynamics
of cellular response to radiation.
Summary
Nagasaki University soft X-ray microprobe has now
offered a unique opportunity to study the mechanism of non-
targeted effects of ionizing radiation. There is also consider-
able interest in the application of LPDS for a wide variety

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of research fields. Our collaborative works are expected to
contribute to the better understandings of the radiation
effects on living organisms.
THE CRIEPI MICROBEAM SOFT X-RAY
IRRADIATION SYSTEM
Introduction
Today, many facilities have been developed and planned
microbeam irradiation systems using charged particle radia-
tions (see in extended abstracts of 8th International Work-
shop: Microbeam probes of Cellular Radiation Response).
However, X-ray microprobes, which have been developed
and operated constantly, were only at the Gray Cancer Ins-
titute (UK)40,47) and at the Photon Factory utilizing SR X-
rays.37,48) Microbeam soft X-ray irradiation system at Central
Research Institute of Electric Power Industry (CRIEPI),
Tokyo, Japan, has been developed in March, 2007 to inves-
tigate cellular response to low dose radiation and non-target-
ed effects, such as radiation-induced bystander responses,
adaptive responses and genomic instability. Our system is
characterized by (1) tabletop (2) X-ray focusing system
using Fresnel zone plate (FZP), and (3) on-line confocal
laser microscope.
The X-ray source and the focusing assembly
Following the Gray Cancer Institute ultrasoft X-ray
microprobe,40,47) Fresnel zone plate (FZP) is used to focus
characteristic X-ray generated by the electron bombardment
of a target. The electron gun is OME-0055LBW (Omega-
tron) with lanthanum hexaboride (LaB6) cathode, which can
generate a high-brightness focused electron beam, and is
operated at voltages up to –30 kV relative to the target (Fig.
9C). The electron beam is focused with electromagnetic lens
onto the surface of the target made of aluminum. Rotary
pumps, turbo molecular pumps and vacuum gauges are set
to the electron gun chamber and the target chamber inde-
pendently. The electron gun is connected with the target
chamber with a gate valve. To keep the LaB6 filament in a
good condition, pressure in the electron gun chamber and
the target chamber is always kept at 10–7–10–8 Torr.
Characteristic K-shell X-ray of aluminum (1.49 keV) is
generated by the focused electron bombardment of an alu-
minum target (Fig. 9D). Hereafter, “soft X-rays” will be
used to denote 1.49 keV X-rays. The bremsstrahlung X-rays
having higher energy, which are also generated together with
characteristic radiation, are removed by reflecting the graz-
ing incidence mirror. This mirror is 20 mm × 10 mm made
of Au (Fig. 9E). The incident angle is 1.5°. The vacuum
window is made of 0.3 mm × 0.3 mm silicon nitride, which
is 150 nm thick (Fig. 9F). The FZP is 150 μm in diameter,
designed and manufactured by NTT-AT Nanofabrication
(Fig. 9G). Exposure period is controlled using the shutter
(Fig. 9H). The order selecting aperture (OSA) is used to
select first-order diffracted soft X-ray by blocking unwanted
zero and higher-order X-rays. The OSA consists of the pin-
hole, 30 μm in diameter (Fig. 9I). From the vacuum window
to the OSA, helium gas is continuously blown to minimize
attenuation of the soft X-ray.
The Cell imaging and irradiation
Autostage, cell irradiation dish and irradiation software
are same as the synchrotron X-ray microbeam irradiation
system at the Photon Factory (PF), High Energy Accelerator
Research Organization (KEK, Ibaraki, Japan).37,48) Cells are
plated on the 1.5 μm-thick Mylar film based cell irradiation
dishes (34 mm in diameter, Fig. 10A).37) We have developed
custom-made stage top incubator (Tokai Hit, Shizuoka,
Japan) that accommodate cell irradiation dish to enable real-
time live cell analysis (Fig. 10B).
High resolution cooled CCD camera (ORCA-ER,
Hamamatsu Photonics) is combined with irradiation system.
To irradiate cells, the beam position is detected using a scin-
tillator and records the coordinates of the center of soft X-
ray microbeam. Cell nuclei are stained with Hoechst 33258
at a concentration of 1 μM for 30 min before irradiation.
After twice wash with PBS, cells are incubated in a fresh
medium for the irradiation. The positions of cell nuclei are
determined by the fluorescent image obtained using the
CCD camera. The position of targets and exposure period
are memorized in the irradiation software. For immunofluo-
rescent study, confocal laser scanning microscope (FV300,
Olympus, Tokyo, Japan) is also combined with irradiation
system.
Characteristics of soft X-ray microbeam
The measurement of energy spectrum indicated that
focused 1.49 keV of Al-Kα X-ray can be acquired at the
sample position through the OSA (data not shown). Beam
size was measured by knife-edge scanning and observed
beam size was 1.8 μm in diameter (Fig. 11). Dose rate in the
irradiated region was about 1 Gy/s under the usual operating
condition.
Detection of DNA damage induced by soft X-ray micro-
beam irradiation
The induction of complex clustered DNA damage,
having multiple DNA lesions within a few helical turns, has
been considered as the cause of efficient cell killing of high
LET charged particle radiations. To examine a possibility of
the induction of complex clustered DNA damage induced by
high dose of focused soft X-ray microbeam irradiation,
human cervical carcinoma HeLa cells were irradiated with
X-ray microbeam for 30 or 60 sec (about 30 or 60 Gy within
irradiated region). Cells were fixed with cold methanol and
rinsed with cold acetone 0.5 or 8 h after irradiation. DNA
lesions induced by soft X-ray microbeam were visualized by
immunofluorescence staining with anti-rabbit 53BP1 and

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Fig. 9. Microbeam X-ray irradiation system of CRIEPI. (A) External view of system. (B) The X-ray microfocus source and
confocal laser scanning microscope. (C) Electron gun and target chamber. (D) Aluminum target. (E) The grazing incidence
mirror. (F) The vacuum window. (G) Fresnel zone plate (FZP). (H) The FZP assembly and the shutter. (I) Order selecting
aperture (OSA).
Fig. 10. Cell irradiation dish. The cell irradiation dish used in this system is same as that in synchrotron X-ray
microbeam irradiation system, Photon Factory, KEK (Ibaraki, Japan). (A) Cell irradiation dish was set on autostage.
(B) The stage incubation chamber.

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anti-mouse phosphorylated histone H2AX (Ser139, general-
ly known as γ-H2AX) antibodies. Co-localization of 53BP1
and γ-H2AX could be clearly observed in the soft X-ray irra-
diated cell nuclei 0.5 h after irradiation (Fig. 12, upper
panels). Localization of DNA repair proteins was disap-
peared within 8 h (data not shown) in cells irradiated with a
few Gy of high energy, wide beam X-rays. On the other
hand, co-localization of 53BP1 and γ-H2AX was not
resolved even 8 h after high dose of X-ray microbeam irra-
diation and was not diffused from irradiated area(Fig. 12,
lower panels), suggesting locally multiple-damaged site, or
clustered DNA damage, may be induced with soft X-ray
microbeam similarly as with high-LET charged particle irra-
diation.
Summary
Table-top microbeam X-ray irradiation system using AlK
X-ray has been developed at the CRIEPI, Tokyo, Japan.
Obtained beam size was 1.8 μm in diameter and dose rate
in the irradiate region was about 1 Gy/s. This system is now
working routinely, and open to outside researchers under
collaborative research.
MONOCHROMATIC X-RAY MICROBEAM
IRRADIATION FACILITY AT THE PHOTON
FACTORY
Introduction
X-ray microbeam irradiation system at the Photon Factory
was designed so as to fully utilize characteristics of synchro-
tron radiation as light source. Synchrotron radiation (SR) is
emitted from high energy electrons generated in electron
storage-type accelerator. Characteristic of SR are strong
intensity in wide energy range from vacuum-ultraviolet to
X-rays, and nearly parallel or directional beam according to
the relativistic effect. Using latter characteristics, X-ray
microbeam can be produced either by simple cutting of
beam with precise slit system or by a focusing system, such
as Kirkpatrick Baez (K-B) mirror system. Both systems have
merit and demerit; in former system, beam size can be
Fig. 11. The output of X-ray was measured by the scanning of
photodiode set on autostage. Beam size could be estimated at 1.8
μm in diameter.
Fig. 12. Localization of 53BP1 and phosphorylated histone H2AX (γ-H2AX) was observed by immunofluores-
cence. HeLa cells were cultured on cell irradiation dish and irradiated with X-ray microbeam for 30 or 60 sec. Cells
were fixed 0.5 h (upper panels) and 8 h (lower panels) after irradiation. Following immunofluorescence, cells were
observed using online confocal laser scanning microscope.

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changed arbitrarily above 5 μm square with constant inten-
sity, in the latter, more intense beam (higher photon density)
is available in smaller beam while change of beam size is not
so easy. Considering these points, we decided to employ a
precise slit system to obtain X-ray microbeam. We have
developed three type of microbeam irradiation system. All
the developed systems have been installed at BL-27B in the
Photon Factory, Institute of Materials Structure Science,
High Energy Accelerator Research Organization (KEK) in
Tsukuba, Japan. Experimental stations at BL-27 are situated
in the biological sample preparation area, where incubators
and other equipments to grow and handle mammalian cells
are available.
Energy-fixed (5.35 keV) X-ray microbeam irradiation
apparatus
First type of the system is composed of three parts.48) The
first part is to produce X-ray microbeam by a high-precision
slit system to cut out the beam. This slit system is set just
below the sample stage. Minimum beam size obtained so far
is 5 μm square by the slit system. Before making a micro-
beam, the beam is reflected right angle upward by diffrac-
tion of Si(311). Due to this process, energy of X-rays is fixed
to 5.35 keV, which can penetrate into tissue nearly half mm
deep. The second is an epi-fluorescent microscope equipped
with a precise motorized stage, on which the sample dish is
horizontally fixed and irradiated with X-ray microbeam. The
third is a fluorescence image analyzer (computer) with a
sensitive CCD camera, which recognizes the target cells and
their positions. It also controls irradiation of X-ray beam to
the targeted cells, one by one, automatically. Intensity of dif-
fracted X-ray beam at the sample position is about 40 R/sec
(ca. 104 photons/s in 10 μm square). Positioning accuracy of
the targets, coordinates of which are automatically analyzed,
are about 1 μm. This system can irradiate 1000 cells per
hour, so that we can keep the cells in a good physiological
condition during the irradiation process. Figure 13 demon-
strates performance of our system. Distribution of immuno-
staining of γ-H2AX clearly depends upon the irradiated
beamsize.
Energy-tunable X-ray microbeam irradiation appa-
ratus
Second system developed is an energy-tunable X-ray
microbeam system. One of the advantages of using SR as
light source is that we can choose any energy of monochro-
matic X-rays with practical intensity. Using this merit we
have a long experience in studying the energy dependence
of X-ray-induced biological effects, including the effects of
inner shell photoabsorption and Auger effects. According to
the recent reports using microbeam, it has been revealed that
radiation-induced signaling in the cell plays an important
role for deciding the final biological effects. In order to study
the signal induction process from energetic viewpoint, we
developed an energy-tunable X-ray microbeam system, by
which we can irradiate with monochromatic X-ray of inner-
shell absorption edges of certain elements. We have to use
the horizontal beam as emitted from the electron storage
ring, hence we need to overcome some difficulties such as
vertical positioning and sample chamber containing cells.
After confirming the accuracy/reproducibility of sample
positioning stage within a few micron, we have studied the
effects of microbeam X-ray tuned to the absorption edges of
endogenous Ca and Fe which is incorporated as phenanthro-
line-Fe.
Irradiation apparatus aiming at cytoplasm
Third one was designed to irradiate cytoplasm only,
avoiding irradiation to cell nuclei. Using the advantage of
our slit system, we have measured dose-survival relation-
ships of V79 cell in two irradiation conditions with clono-
genic assay.49) One is to irradiate with 10 μm square beam
aiming at nucleus only, the other with 50 μm square aiming
at whole cell. This work revealed that hypersensitivity in low
dose region is more enhanced in nucleus-irradiated cells
than in whole-cell irradiated cells. These results suggest that
intracellular communication between nucleus and cytoplasm
plays an important role in determining the cell death in low
dose region. For further investigation of intracellular com-
munication in irradiated cells, we have developed a system
to irradiate cytoplasm only without irradiating cell nucleus.
In order to shield the nucleus from irradiated in the uniform
irradiation field, we made a gold mask, 15 μm in diameter
and 20 μm thick, on a very thin (200 nm thick) SiN film. The
thickness of the gold was determined to decrease the inten-
sity of 5.35 keV X-rays to less than one-thousandth. SiN
film is nearly transparent (more than 99% transmission) to
this energy of X-rays. SEM image of this mask is shown in
Fig. 14. The mask was mounted on a small X-Y stage and
set in the X-ray path between the slit system and the sample
stage. Using the scintillator dish to observe the shape and
Fig. 13. Immunostaining (green) of γ-H2AX in cell nuclei (red)
irradiated with microbeam of different sizes.

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position of the X-ray beam, we adjusted the position of the
mask and the size of the beam. Finally, we got a 50 μm by
50 μm beam with uniform intensity, at the center of which
located was dark area of 15 μm diameter by the gold mask.
Image of the beam taken as fluorescent intensity of scintil-
lator is shown in Fig. 15.
When we want to irradiate only cytoplasm of the cells,
center of the mask was recognized as the beam position and
cytoplasm of cells, nuclei of which were stained with Hoe-
chst dye and recognized by its fluorescence, were irradiated
with X-rays, leaving nucleus unirradiated. Survival data are
now being accumulated and survival curve of cytoplasm-
irradiated cells will soon be presented.
Summary
Three types of X-ray microbeam irradiation apparatus
have been developed and now working routinely using syn-
chrotron X-rays at the Photon Factory, KEK, Tsukuba,
Japan. Energy range available now is from 4 keV up to 20
keV. Beam size can be changed easily from 5 μm square or
above.
DEVELOPMENT OF THE CAS- LIBB SINGLE-
PARTICLE MICROBEAM
Introduction
The research team in the Key Laboratory of Ion Beam
Bioengineering, Chinese Academy of Sciences (CAS-
LIBB), in Hefei, China, proposed a Single Particle Micro-
beam (SPM) program for radio-biological irradiation in
1991, and put forward a further project of a SPM in 1995.
In 2002, a high-quality SPM facility was installed. After two
years of adjustment and optimization, the SPM facility has
achieved perfect performance. Thereby, an ideal research
platform is provided for studies on risk assessment on low-
dose environmental exposures, genomic instability in cells,
“bystander effects” and the underlying mechanisms of radi-
ation damages in living cells, etc..50)
Facility components and modules
The CAS-LIBB microbeam facility is comprised of these
elements: 1) a 5.5-MV Van de Graaff accelerator, 2) two
deflecting magnets for producing mono-energetic charged
particles beam, 3) the beam-transporting pipeline with feed-
back devices, magnetic quadrupoles, diaphragms and
vacuum bumps, and 4) electric beam shutter and the micro-
beam collimator at the end-station. Figure 16 shows the
beam line layout of the facility. Ions produced from a radio-
frequency (RF) ion source are accelerated to an energy in the
range of 2.0–3.0 MeV and transported to the first beam-
deflecting magnet through a beam guider and a pair of col-
limation slits. After the first deflection, the mono-energetic
beam goes forward horizontally through the beam-stabilizer,
quadrupole magnets and the beam shutter plates, and then
enters the second beam-deflecting magnet. The up-going
Fig. 14. SEM image of the mask. (provided by NTT-AT).
Fig. 15. Shape of the X-ray microbeam aiming at cytoplasm only.
Fig. 16. Beam line layout of the single-particle microbeam fac-
ility.