Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 47 (2014)

Observation of unrepairable lesions in DNA by using 3-MeV
proton microbeams produced by glass capillaries
T. Ikeda,＊1 M. Izumi,＊1 V. Mäckel,＊2 T. Kobayashi,＊2 K. Ogiwara,＊2 T. Hirano,＊3 Y. Yamazaki,＊2 and T. Abe＊1,＊3Microbeams allow extremely efficient alteration of ordamage to a small region in the target with minimum beam
intensity. To date, X-ray, UV, and visible light laser havebeen used as microbeams. However, to create double strand
breaks (DSBs) in DNA, which are difficult to repair,multiple photons are required and have to be focused on theDNA. Because of the finite size of the focal spot, the beam
intensity needs to be high. This intense beam creates
multiple DSBs. In contrast, when ion beams are used, asingle ion can create a DSB via ballistic electrons andradicals generated along the ion’s trajectory. This is anadvantage of ion-beam irradiation. We developed a method
involving ion-microbeam irradiation to single cells inculture using tapered glass capillaries with outlet diametersof ~1 m.1,2) The capillary is known as glass pipet for
microinjection or a glass electrode for real timemeasurements of the voltage potential within the neurons.
The number of ions irradiated to single cells is controlled bya pulsed beam. However, some DSBs can be repaired
quickly even if ions hit the DNA. Here, we measured thefluorescence brightness corresponding to unrepaired DSBs
after irradiation for different numbers of ions input per cellto confirm that a small number of protons can cause
unrepairable damage.
The beam used for irradiation was composed of 3-MeV
protons that were generated by a RIKEN Pelletron
accelerator and transported to the cell irradiation port,
which uses an inverted microscope (OLYMPUS IX-71). A
glass capillary optics system with a thin plastic end-window(diameter, 2 m) was mounted at the beam port at 45o sothat a petri dish filled with solution can be used. The
capillary tip can go close to the cells which is advantageous
for suppressing multiple scattering of ions before they reachthe target. To adjust the number of protons, which is
proportional to the dose, the time window of the beam pulse,which included a maximum of 10 ions, was set at 1 - 5 s,
wherein the number of ions in a pulse follows Poisson(b)100
LET (keV/ m)(a)distribution. We irradiated 50-1,000 ions per cell to reduce
statistical error.
Figure 1(a) is a microscopic view of HeLa cells during
irradiation. Cell nuclei marked by yellow circles wereselected. The capillary tip, which was above the cells, is at
the lower left in Fig. 1(a). It took ~20 min to irradiate 60
cells in one dish. After the irradiation, cells grown inglass-bottom dishes were washed three times 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-40 in PBS at 4°Cfor 5 min, and phosphorylated histone H2AX was detectedby using rabbit antibody (Millipore) and an
Alexa488-conjugated donkey anti-rabbit IgG (JacksonImmunoResearch Laboratories).
We obtained clear fluorescence distributions only in theirradiated nuclei. The brightness of the foci (determined interms of Alexa488 fluorescence) in each irradiated nucleus
was proportional to the number of unrepaired lesions in
DNA at the time cell fixation was initiated. The brightness
data were displayed in a histogram by using the software,
Image-J .3) After subtracting the low background level offoci seen for the non-irradiated cells, histogram integration
was compared according to the microbeam intensities[Table 1]. The brightness increased with but was notproportional to microbeam intensity, perhaps because ofsaturated damage due to the large number of ions.
The linear energy transfer (LET) of 3-MeV proton was~10 keV/m [Fig. 1(b)]. When heavier ion pieces are
selected, higher LETs will be obtained. Moreover, the ion
stopping positions can be localized at a certain depth insidea cell because of their short ranges. In this case, extremely
high energy deposition corresponding to the Bragg peak isavailable for destroying a single cell or a small region oftissue.
Table 1. Integrated brightness of the foci.
Microbeam
Brightness of Alexa488

intensity (ions/cell)
50200
1,00080
6040(arbitrary unit)
733875940

20001234

Proton energy (MeV)

References

51) Y. Iwai, T. Ikeda, T. M. Kojima, Y. Yamazaki, K. Maeshima, N.
Imamoto, T. Kobayashi, T. Nebiki, T. Narusawa, and G. P.
Pokhil: Appl. Phys. Lett. 92, 023509 (2008).
2) V. Mäckel, W. Meissl, T. Ikeda, M. Clever, E. Meissl, T.
Kobayashi, T. M. Kojima, N. Imamoto, K. Ogiwara, and Y.
Yamazaki: Rev. Sci. Instrum. 85, 014302 (2014).
3) Image-J 1.47v, downloaded from http://rsbweb.nih.gov/ij/Fig. 1. (a) HeLa cells (yellow circles) and the tip of theglass capillary, (b) LET in water vs. proton energy.
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