Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 53

Ion-microbeam irradiation to damage DNA in RPE cell nucleus withtapered glass capillary optics at Pelletron facility
T. Ikeda,∗1 M. Sakai,∗2,∗1 K. Takemoto,∗2,∗1 A. Shibata,∗3 M. Izumi,∗1 and M. Uesaka∗2Cell irradiation experiments with ion microbeams havestarted at a new beamline of the RIKEN Pelletron accelerator facility in the Nishina R&D Building. The purpose of the experiments is to investigate the dynamicsof the accumulation of restoration proteins after DNAdouble-strand breaks (DSB). The microbeam irradiation
allows artiﬁcial DNA damage, which deﬁnes the time
zero for the accumulation of repair proteins. To create DSB eﬃciently, ions with high linear energy transfer(LET) should be used. Figure 1 shows the LET in waterfor H and He ions with an energy E below 5 MeV. The H
and He ions have maximum LETs of 80 and 240 keV/µm,respectively, just before stopping. If we use He ions withan energy of 4.5 MeV, an LET of more than 100 keV/µm
is available through an ion range of 27 µm. The LET
value is large enough to create DSB.
The irradiation was performed at the BL-W30 beamline employing a single tapered glass capillary optics atthe beam port, where the microbeam was emitted in thehorizontal direction. A He-ion beam was accelerated to4.5 MeV with a tandem accelerator (Pelletron 5SDH-2,1.7 MV max.), and it entered a glass capillary having
an outlet diameter and end window thickness of 11.4 µm
and 8.6 µm, respectively. The details of the capillary microbeam at an old beamline of our facility are reportedelsewhere.1,2) The target was nuclei of retinal pigment
epithelial (RPE) cells cultivated in a liquid medium. Thecells were fully conﬂuent in petri dishes, the glass bottoms of which were marked to ﬁnd irradiated cells for theﬂuorescence observation after the irradiation. As shownin Fig. 2(a), the petri dish was positioned at an angle ofapproximately 45◦ to the capillary axis. To maintain thecells in the dish ﬁlled with the medium, it was dripped
onto the bottom of the dish at a speed controlled bya peristaltic pump. The microbeam intensity was set
to approximately 15 ions/s, which was conﬁrmed before
and after the irradiation. The irradiation distance be-Fig. 1. Comparison of the LETs of H and He ions in waterwith an energy between 0 and 5 MeV.
∗1
∗2
∗3

RIKEN Nishina Center
The Graduate School of Engineering, Univ. of Tokyo
Gunma University Initiative for Advanced Research (GIAR),
Gunma Univ.

Fig. 2. (a) Cell irradiation with a glass capillary. The angle
was set to approximately 45◦ with respect to the capillaryaxis. (b) Irradiated RPE cell nucleus with DSB points.

tween the capillary outlet and the cells was set to several
micrometers. The distance was always monitored by amicroscope above the irradiation point. Each dish wasmoved for 3–8 min to irradiate the cells on a line acrossthe dish. The total number of dishes was four. Afterthe irradiation, 30 min was spent for the repair process
based on an enzymatic reaction. Subsequently, the cells
were ﬁxed with 3% paraformaldehyde (PFA)-2% sucrosein phosphate-buﬀered saline (PBS), followed by washingwith PBS. Next, the dishes were ﬁlled with PBS for delivery to Gunma University for observation by an optical
microscope with three-dimensional structured illumination, 3D-SIM, microscopy (DeltaVision OMX version 4,GE Healthcare).
Figure 2(b) shows a bottom view of one of the irradiated cells. The blue area corresponds to the cell nucleus labeled by DAPI. The bright blue points are DSB
positions where phosphorylated histone H2AX was laR 488 by the immunoﬂuorescence
beled with Alexa Fluor⃝
method. The DSB points are clearly aligned on a linewith extremely low background ﬂuorescence. The line is
expected to be a He ion track stopping at or penetrating
the nucleus. The photo is one of the z-sliced photos witha slice step ∆z of 0.25 µm.
The brightness information of each pixel in the images
was stored as a 3D map. Using the map, the directionsof all ion tracks and the density distributions of DSB andother restoration proteins were extracted. The time evolution of repair of DSB as a function of the distance fromthe ion track will be obtained in the next experiment bychanging the time between irradiation and ﬁxing.
References

1) V. Mäckel et al., Rev. Sci. Instrum. 85, 014302 (2014).
2) T. Ikeda et al., Quantum Beam Sci. 4, 22 (2020).

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