JAEA-Review-2010-065.pdf:15.99MB - 日本原子力研究開発機構

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Target Irradiation of Individual Cells Using Focusing
Heavy-Ion Microbeam of JAEA-Takasaki
T. Funayama, T. Sakashita, Y. Yokota and Y. Kobayashi
Heavy-ion beam is utilized for heavy-ion cancer therapy
and ion beam breeding because of its unique biological
effectiveness. However the elucidation of mechanisms
underlying biological response of heavy-ion radiation is
necessary to advance these useful applications. Localized
irradiation of specific regions within organisms using
heavy-ion microbeam systems provides an attractive means
of investigating the mechanism of heavy-ion radiation action.
Therefore, we had developed the heavy-ion collimating
microbeam system at the facility of Takasaki Ion
Accelerator for Advanced Radiation Application (TIARA)
of the Japan Atomic Energy Agency (JAEA), and utilized
for analyzing heavy-ion induced biological effects 1) .
However, there is a difficulty in generating finer beam
that is capable for carrying out precise subcellular irradiation
in our current system, because of inevitable scattering of
ions at the edge of micro collimator. The scattering ions do
not hit on the targeted cells, thus made mishit cells in the
sample. On the other hand, there are little scattering ions
when microbeam is generated by focusing system using
magnetic lens, thus the system is expected to target and
irradiate cells accurately. Therefore, we developed new
focusing microbeam line at another vertical beam line of
2)
AVF cyclotron of TIARA, JAEA .
New system can focus heavy-ion beam to minimum one
micrometer in vacuum using a quadruplet quadrupole lens
system, and equipped with an X, Y beam scanner for fast
hitting of single ion to micron scaled samples. To irradiate
this finer microbeam to the specific region of individual
cells, a cell targeting system was designed and installed
under the beam extraction window. The system was
consisted of inverted microscope and automatic stages of
7 axes. The system can be controlled completely from
remote preparation room. For avoiding vibration, the
system is installed on the rigid frame that is hanged and
fixed to the magnetic lens. At the bottom port of the
microscope, a high sensitivity cooled CCD camera was
installed for detecting weak fluorescence of scintillator and
stained target cells.
The position of focusing microbeam was detected under
microscopic view using CaF2(Eu) scintillator. The ions
irradiated to the sample were detected by solid state ion
detector installed on the objective revolver of the
microscope, and counted by counter NIM module to control
a fast beam shutter.
Using the system, irradiation of HeLa cells were carried
out. The cytoplasm of cells were stained with CellTracker
Orange fluorescent dye (Invitrogen) and inoculated on a film
of ion track detector, CR39, of 100 µm thick. For avoid
JAEA-Review 2010-065
Radiation-Applied Biology Division, QuBS, JAEA
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drying, the sample was covered by Kapton film of 8 µm in
thick and sealed by petrolatum. The positions of each
target cell were extracted from the fluorescent cell image
using self-developed image analysis code, which is
optimized for analyzing CellTracker-stained cell image
(Fig. 1). The cells were, thereafter, targeted and irradiated
with focusing Ne ion beam (13.0 MeV/u, LET =
380 keV/µm). The irradiation was carried out by moving
the cells to the position of beam spot using automatic stage
system. The ions were irradiated from up above, and the
number of ion irradiated was counted by solid stage detected
installed under sample stage.
After irradiation, the tracks of traversed ion were
visualized by etching of CR39 film and hit positions of
irradiated ion were confirmed. The ions were well focused
and hit on targeted cells precisely. Therefore, we
concluded that the new system can target and irradiate
individual cultured cells by focusing heavy-ion microbeam.
Fig. 1 Detection of cell target position using image
analysis code optimized for CellTracker-stained cell
image.
References
1) T. Funayama et al., J. Radiat. Res. 49 (2008) 71-82.
2) M. Oikawa et al., Nucl. Instrum. Meth. Phys. Res. B 260
(2007) 85-90.