Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 48 (2015)

Ion track observation in cell nucleus irradiated by 3 MeV He ion microbeams
produced with glass capillaries
T. Ikeda,＊1 M. Izumi,＊1 V. Mäckel,＊2 T. Kobayashi,＊2 R. J. Bereczky,＊1 T. Hirano,＊1 Y. Yamazaki,＊2 and T. Abe＊1Microbeam irradiation of cultured single cells using
tapered glass capillaries with outlet diameters of the orderof 1 m has been performed employing RIKEN Pelletron
accelerator. Apparatus with a 3-m outlet diameter isreported in elsewhere1). Microbeam is a unique scheme 2)that can actively select an irradiation volume of ~m3.
When a microbeam of He ions of a few MeV hits a target,e.g., a biological cell, the ions will stop at the depth
corresponding to their range (10~20 m). Any other parts
downstream of the stopping volume will not be damaged.
This is one of advantages of employing beams of a few
MeV. The glass capillaries with thin end-windows play animportant role in delivering the ions directly to the cells insolution.
At RIKEN, we have developed a unique technology formutation breeding using high-energy heavy-ion beams fromRIKEN Ring Cyclotron. Such high linear energy transfer(LET) ions produce clustered DNA damage that cannot berepaired by the cell itself, leading to cell death, or can only
be repaired incompletely by the cell, which induces
mutations. In order to investigate both the lethality and theeffectiveness of mutation induction, the position selectivityof the microbeam will be needed because hitting ofdifferent parts may cause unwanted effect. Here wedemonstrate the DNA damages along ion tracks in a cell
nucleus using a microbeam with relatively high LET.
A 3-MeV He ion microbeam was used for the irradiationof the nucleus of human cells (HeLa cells) because it is
known that there is a similarity in the radiation response
between plant and animal cells. The ions were generated bythe Pelletron and transported to the cell irradiation port1). A
5-cm-long glass capillary optics with a 4-m-thickend-window whose diameter was 3 m and with an inlet
diameter of 0.8 mm was installed at the beam port with anangle of 45o with respect to the horizontal plane so thatwidely-used petri dish filled with liquid solution can beused. The LET of a 3-MeV He ion is 150 keV/m in water.
The range of the ions is only 12 m in water after theend-window. In order to adjust the dose of the irradiation toeach cell, a beam chopper consisting of an electrostatic
beam deflector was employed so that a few to ten ions were
included in a short pulse of 0.8 s. The pulse was repeated100-1000 times according to the number of the required
ions to the target.
It took approximately 12 min to irradiate about 40 cells in
one dish, and totally 168 cells in 4 dishes were irradiated.

After the irradiation, the Double Strand Breaks (DSBs) at
DNAs were fluorescent-labeled as follows. The time for therepair process based on an enzymatic reaction was 20 min
after the irradiation, and then the cells were washed threetimes with ice-cold phosphate-buffered saline (PBS) and
fixed with 4% formaldehyde in PBS at 4°C for 20 min.
Then, the cells were permeabilized with 0.5% Nonidet P-40in PBS at 4°C for 5 min, and phosphorylated histone H2AX
was detected by using rabbit antibody (Millipore) and an
Alexa488-conjugated donkey anti-rabbit IgG (JacksonImmunoResearch Laboratories).
Figures 1(a) and (b) are the cross-sectional and bottom
views of the irradiated cells, respectively, reconstructedfrom the photos taken by changing the focus position along
the z-axis (vertical axis). Cell nuclei are identified as
bean-shaped blue regions with a width of ~15 m and havefluorescence from a stain DAPI binding to A-T rich regionsin DNA. The outlines of the cells are not seen. The DSBs
are detected as bright points with green color from
Alexa488. Even without irradiation, DSBs can take place asan activity of a living cell. However, the concentration ofDSB bright points along a line is an evidence of artificiallesion. The ion track with the angle of 45o is clearly seen as
a fluorescent line in Fig. 1(a). We succeeded in observationof a visible track for MeV ions inside a cell nucleus. It isconfirmed that MeV-ion irradiation made DSB lesions inthe DNA, which may cause gene defects. Furtherexperiments considering other conditions, e.g., LET, controlof cell cycle, and so on are needed as well as the number ofions necessary to form a track.

Fig. 1. Fluorescent lines corresponding to the ion track in ahuman cell nucleus. (a) Cross-sectional view of theirradiated cells at the horizontal line in the bottom view. (b)Bottom view of the cell.
References

RIKEN Nishina Center＊2
Atomic Physics Laboratory, RIKEN＊11) V. Mäckel et al.: Rev. Sci. Instrum. 85, 014302 (2014).
2) Y. Iwai et al.: Appl. Phys. Lett. 92, 023509 (2008).

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