- Email: [email protected]
Biological studies using mammalian cell lines and the current status of the microbeam irradiation system, SPICE
Nuclear Instruments and Methods in Physics Research B 267 (2009) 2171–2175
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Biological studies using mammalian cell lines and the current status of the microbeam irradiation system, SPICE T. Konishi a,*, T. Ishikawa a, H. Iso a,b, N. Yasuda a, M. Oikawa a, Y. Higuchi a,b, T. Kato a,c, K. Hafer d, K. Kodama a,b, T. Hamano a, N. Suya a, H. Imaseki a a
Dept. of Technical Support and Development, Fundamental Technology Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Neos-Tech Co. Ltd., Benten 4-11-13-202, Chuo-ku, Chiba 206-0045, Japan c Graduate School of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshimaku, Tokyo 171-8501, Japan d Department of Radiation Oncology, UCLA School of Medicine, Los Angeles, CA, USA b
a r t i c l e
i n f o
Available online 13 March 2009 PACS: 29.27.Eg 29.27. a 61.80. x 87.50.Gi 87.80. y Keywords: SPICE Microbeam Protons Mammalian cells CR-39 Biological effect
a b s t r a c t The development of SPICE (single-particle irradiation system to cell), a microbeam irradiation system, has been completed at the National Institute of Radiological Sciences (NIRS). The beam size has been improved to approximately 5 lm in diameter, and the cell targeting system can irradiate up to 400– 500 cells per minute. Two cell dishes have been specially designed: one a Si3N4 plate (2.5 mm 2.5 mm area with 1 lm thickness) supported by a 7.5 mm 7.5 mm frame of 200 lm thickness, and the other a Mylar film stretched by pressing with a metal ring. Both dish types may be placed on a voice coil stage equipped on the cell targeting system, which includes a fluorescent microscope and a CCD camera for capturing cell images. This microscope system captures images of dyed cell nuclei, computes the location coordinates of individual cells, and synchronizes this with the voice coil motor stage and single-particle irradiation system consisting of a scintillation counter and a beam deflector. Irradiation of selected cells with a programmable number of protons is now automatable. We employed the simultaneous detection method for visualizing the position of mammalian cells and proton traversal through CR-39 to determine whether the targeted cells are actually irradiated. An immuno-assay was also performed against c-H2AX, to confirm the induction of DNA double-strand breaks in the target cells. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Single-cell microbeam irradiation systems have recently become important tools for radiation biology. The major characteristic of microbeam irradiation systems is a very narrow beam of radiation, micrometer size or smaller, corresponding to cellular or sub-cellular dimensions. In addition, microbeam irradiation of exactly one to any preset number of particles per cell allows study of radiation risks from very low doses to be addressed. Moreover, microbeam techniques can be used to address such questions as the effects of irradiation on unirradiated neighboring cells, such as the bystander effects, and the relative sensitivities of different parts of the cell. Multiple microbeam facilities have been developed for biological research [1–13]. In Japan an array of microbeam facilities with different radiation sources is available for biological studies [14–16]. Such facilities have been used to study low-dose exposure, hyper radio-sensitivity, bystander effects and others [17–21]. * Corresponding author. Tel.: +81 43 206 3032; fax: +81 43 206 3514. E-mail address: [email protected] (T. Konishi). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.03.060
The single-particle irradiation system to cell, SPICE, at the National Institute of Radiological Sciences (NIRS) generates 3.4 MeV protons with an approximately 5 lm diameter beam, and is the only proton microbeam irradiation system in Japan [16]. SPICE is currently operational for biological studies. This paper describes the results of improvements of the beam line, along with preliminary data from biological experimentation. 2. Materials and methods 2.1. Outline of the microbeam irradiation system, SPICE The details of SPICE have already been described [16]. Briefly, the electrostatic accelerator facility of NIRS supplies protons and helium ions by a Tandetron accelerator (HVEE, High Voltage Engineering Europe Ltd.) [22]. Three horizontal beam lines are available for PIXE analysis. PIXE Analysis System and Tandetron Accelerator, PASTA, consists of a conventional PIXE line for analysis under vacuum conditions, an ‘‘in-air” PIXE line [23], and a microbeam scanning PIXE line for two-dimensional mapping of multi-elemental distributions [24]. The fourth beam line, SPICE is a vertical beam
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T. Konishi et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2171–2175
line which diverges from the microbeam scanning PIXE (microPIXE) line and transported upward by a 90° bending magnet installed in the middle of the micro-PIXE beam line. This vertical beam is focused by a mono-bloc triplet quadrupole lens (Q lens; OM170, Oxford microbeams, Ltd.) to exclude such low-energy particle components by scattering seen in microbeams produced by the collimation method [1]. 3.4 MeV protons extracted into atmosphere are used to irradiate cells. Because the beam diameter deteriorates outside of the vacuum system due to proton scattering in air, the distance the beam travels through air must be minimized. In our previous paper, we used Kapton film as a beam exit window. This film bends more than 300 lm toward the vacuum and this air gap prevents the beam from being focused to less than 10 lm in diameter. In this work, a 1 lm thick Si3N4 membrane (Silcon Ltd.) of 2.5 mm 2.5 mm area with a 200 lm thick frame of 7.5 mm 7.5 mm area was glued onto the beam exit pipe. A schematic diagram of the beam exit window and the configuration of the surrounding components are shown in Fig. 1(A). The air gap between the Mylar film of the cell dish and the beam exit window was set to be below 100 lm to keep the beam as focused as possible prior to reaching the cell position. The bending of the Si3N4 membrane is approximately 40–50 lm. The air gap was determined by measuring the
difference in operating distance of a 20 objective lens from the focused position at the surface of beam exit window to the surface of the Mylar film of the cell dish. It was adjusted by inserting a metal thin foil of suitable thickness between the stage and the steel plate of the cell dish. For the routine irradiation of cells, the beam intensity can be controlled to be less than 1.0 105 protons per second by adjusting the objective slits installed in the horizontal beam line. The number of protons traveling through the cells was counted using a scintillation detector set above the cell dish, as shown in Fig. 1. A fast beam deflector system (10 MHz) was installed upstream of the 90° bending magnet and was used to switch the beam off on demand. It consists of two parallel metal electrodes connected to a high-voltage generator. After the scintillation detector detects the preset number of protons that traversed through the CR-39 (or the cells), the computer that controls the voice coil motor stage gives a high-speed trigger pulse to the beam deflector which drops the voltage of one of the parallel electrodes to keep the beam from being transported into the vertical beam line. The voice coil motor stage is self-developed with a Technohands, Co., Ltd. (Yokohama, Japan), and has 10 mm 10 mm traveling range with a position accuracy of 40 nm. By repeating this procedure, it is possible to irradiate a given cell with a programmable number of protons. 2.2. Cell dish and microscope Two types of cell dishes were specially designed. Type one, previously described in [16], is a Si3N4 plate (7.5 7.5 mm frame) attached to a metal dish with Vaseline (Wako, Osaka). The cells are cultured on a 2.5 mm 2.5 mm area of 1 lm thickness, which minimizes the energy loss of protons before they reach the cells. A second type of dish was designed for two reasons: firstly, to culture a larger number of cells and secondly to reduce the air gap between the Mylar film to which the cells are attached and the beam exit window. Cells may be cultured on a 30 mm diameter area of a Mylar film stretched across a 30 mm diameter steel ring to 33 mm diameter hole, with the film in between. A photograph of this cell dish is shown in Fig. 1(B). It is placed on the voice coil stage of the cell targeting system, which also contains a fluorescent microscope (BX51, Olympus) and a CCD camera (ORCA-ER, Hamamatsu). An example of a fluorescent image of the cell nuclei is shown in Fig. 2, which displays the fluorescence of CHO-K1 cells that were dyed with
Fig. 1. Panel A is a schematic diagram of the end station of SPICE, and B is a photograph of the Mylar film cell dish.
Fig. 2. Fluorescent microscope image of CHO-K1 cells dyed with Hoechst 33258. Each cell nuclei is given a reference number for identification in subsequent biological experiments.
T. Konishi et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2171–2175
Hoechst 33258 (Dojindo Laboratories, Kumamoto). Green ellipse shows results of automated cell recognition, which is a selfdeveloped algorithm based on a least-square technique for a second order poly-nominal function [25]. With this recognition algorithm, the X–Y coordinates of individual cells are obtained from the fluorescence signal of each nucleus. Each nucleus was given a number (shown in green) to identify the cells later in biological experiments. Therefore, as according to the sample-preparation protocol, cells were cultured in media containing 1 lM Hoechst 33258 for 2–3 h to stain the nuclei sufficiently for cell recognition from the fluorescent image. The voice coil motor stage is synchronized with a single-particle irradiation system described in the previous section. 2.3. Beam-size measurement using CR-39 The actual beam size was determined by irradiating a 20 mm diameter size and 110 lm thick plastic track detector, CR-39 (HARZLAS TD-1, Fukuvi chemical industry), adhered to the Mylar surface of a cell dish. Fig. 3 shows the irradiated CR-39, etched in 7 M NaOH at 70° for 2 h prior to capturing the image with a confocal laser microscope (Fluoview1000, Olympus). Fig. 3(A) shows the logo mark of the NIRS on the CR-39. Each position was irradiated with 10 protons for a total of 2704 positions with a 10 lm pitch. Panel B shows an expanded image of the area, B, shown in panel A. The irradiation was performed automatically according to an input text file that contains the desired preset number of protons and the X–Y coordinates of the target positions. This system enables one to irradiate 6–8 positions per second, thus the irradiation of the CR-39 shown
Fig. 3. Panel A shows the logo of the National Institute of Radiological Sciences, NIRS on the CR-39. Each position was irradiated with 10 protons for a total of 2704 positions at 10 lm pitch. Bar size, 100 lm. Panel B shows an expanded image of the area, B, shown in panel A. Bar size, 20 lm. Panel C shows the beam size of 20, 50, 100, 150, 200 protons.
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in Fig. 3(A) was completed in less than 10 min. Panel C shows a CR-39 irradiated with 20, 50, 100, 150, 200 protons per position with a 50 lm pitch. The beam size for 200 protons was approximately 5 lm in diameter.
3. Preliminary biological results 3.1. Simultaneous detection of the beam and a cell nucleus We employed a contact microscopy technique [26], previously developed for imaging boron in boron neutron capture therapy to determine whether the targeted HeLa cells (human cervix carcinoma cell line, purchased from Japanese Collection of Research Bioresources Cell Bank, JCRB) are actually irradiated by SPICE. The method is based on the fact that etching speed of CR-39 is enhanced by UV exposure [27]. It is applied to a contact UV microscopy technique proposed by Amemiya et al., in which a relief image of a slice of rat tissue attached to the CR-39 surface and etch pits by neutron-induced alpha-particles were simultaneously recorded on the CR-39 surface [26,28]. This method enables visualization of mammalian cells as a relief on a CR-39 simultaneously with the etch pits that indicate the positions of ion traversals. This technique is very convenient, and enables one to simultaneously obtain geometric information of the cells and ion traversals using only common laboratory equipment, such as a conventional optical microscope, a UV lamp, and commercially available CR-39. HeLa cells were cultured on the surface of CR-39 approximately 24 h before irradiation. Culture conditions were described previously [16,29]. Cells were stained with 1 lM Hoechst 33258 before being irradiated to determine the cell positions in the dish (see Fig. 4(C)). The 3.4 MeV protons traversed the 100 lm air gap, 110 lm of CR-39, the cell nucleus, and finally reached the scintillation counter. After irradiation, the cells were fixed with ice-cooled methanol:acetone = 1/1 solution for 10 min, and rinsed with distilled water. They were then dried for a day in air. The CR-39 plate was exposed to a total dose of 1.6 105 J/m2 of a UV light (mainly 254 nm) with a low-pressure mercury lamp (GL-10, Toshiba) for 4 h. The cells were removed from the CR-39 film, which was then etched in 7 M NaOH for 2 h to detect the etch pits and the embossment of the cell relief. Fig. 4(A) shows an image of HeLa cells as an embossment on the CR-39 surface. Panel A shows an embossment on the CR-39 of the non-targeted cells and the targeted cells, which were irradiated with 500 protons. The targeted cells are indicated by the arrows in panel A, and panel B is a magnified image of B in panel A. The etch pits produced by protons can be clearly identified as well as the cell nuclei on CR-39, showing that targeted cells were actually traversed by the protons. Panel C shows the fluorescence of the cell nuclei. The beam size (etch pits) seen in Fig. 4, is the size of outgoing beam from the 110 lm thick CR-39. The beam size of entering side of CR-39 was approximately 3–4 time smaller in diameter compared to those seen on the outgoing size. This is because of the proton scatter during traversal through the CR-39. Although the etch pits were not detected in the area surrounding the cell nuclei, this was not due to the etching process. If protons traversed somewhere outside of the cell area, larger size etch pits than those in the cell relief should be identified because of the enhanced etching velocity of CR-39 by the previous UV exposure [25–27]. Therefore, we estimated that the beam was targeted specifically to the cell nucleus, although we cannot exclude the possibility that the whole cell area was irradiated. Thus further statistical measurements for beam targeting accuracy are necessary.
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Fig. 4. (A) Microscope overview image of HeLa cell positions as an embossment on the CR-39 surface. (B) In enlarged view of the area indicated in A, where the irradiated cells are indicated by arrows. (C) Displays the fluorescence image captured from area C. Bar size, 50 lm.
Fig. 5. Panel A represents an image of cell nuclei stained by Hoechst 33258, and panel B is a fluorescent image of c-H2AX. Those number shown in green in panel A is the identification number for the individual cell nucleus. Panels A and B are merged in panel C, showing the nuclei in violet, and c-H2AX in green. The number of protons irradiated to targeted cell nucleus is indicated by the number shown in panel C. Bar size, 20 lm. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Immuno-assay for the detection of induced DNA double-strand breaks in targeted cell nuclei The purpose of this preliminary experiment was to determine whether DNA double-strand breaks (DSBs) were induced in the targeted cell. We visualized the DSBs induced by the traversals of protons through the cell nucleus using phosphorlylated histone,
H2AX (c-H2AX), as a marker for DNA DSBs [29,30]. CHO-K1 cells were prepared and cultured on the cell dish 24 h before irradiation. Approximately, 2 105 cells were inoculated on the Mylar film. After staining the cells with Hoechst 33258, the cell dish was set on the voice coil motor stage for image capturing and cell recognition. Targeted cells were selected according to the numbers given in the image (Fig. 5(A)). The number of protons to be irradiated
T. Konishi et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 2171–2175
to those targeted cells was preset, as shown in Fig. 5. After irradiation, the cells were incubated at 37° with 5% CO2/95% air for 30 min for the phosphorylation of H2AX to reach its maximum and then the cells were fixed with 2 ml of 4% paraformaldehyde for 15 min at room temperature. An immuno-fluorescent assay was held using the anti-c-H2AX antibody (CHEMICON International, Inc) and secondary anti-rabbit Alexa 488 antibodies (Molecular Probes), as mentioned elsewhere [16,28]. Fig. 5(B) shows the fluorescence of immuno-stained c-H2AX. Fig. 5(C) is a merged view of panel A and B. The fluorescence of c-H2AX was identified in all the targeted cells, and not in the non-targeted cell nuclei. In addition, the fluorescence increased corresponding to the number of protons irradiated. As a result, the image obtained by UV imaging technique (Fig. 5) and the immuno-stained image of cH2AX (Fig. 5) are the evidences that the cells were targeted accurately. 4. Conclusion Improvements on the microbeam irradiation system, SPICE, have been made. Approximately, 5 lm in beam diameter can be achieved. For cell irradiation, the X–Y coordinates of cells in a dish can be calculated according to the fluorescent cell nuclei imaged by fluorescent microscope. The irradiation procedures can be automatically performed and the maximum speed for cell irradiation is 400–500 cells per minute. The targeting accuracy was checked by employing the simultaneous detection method, which visualized HeLa cells as embossment reliefs and beam position as etch pits on the surface of CR-39. Also CHO-K1 cells were irradiated and immuno-stained against c-H2AX to see an induction of DNA double-strand breaks in the targeted cells. Both experiments showed that SPICE is operational for biological experiments. Further development is underway in order to increase the performance of the microbeam irradiation system to perform studies targeting cell nuclei. References [1] M. Folkard, B. Vojnovic, K.M. Prise, A.G. Bowey, R.J. Locke, G. Schettino, B.D. Micheal, Int. Radiat. Biol. 72 (1997) 375. [2] G. Randers-Pehrson, C.R. Geard, G. Johnson, C.D. Elliston, D.J. Brenner, Radiat. Res. 156 (2001) 210. [3] M. Folkard, G. Schettino, B. Vojnovic, S. Gilchrist, A.G. Michette, S.J. Pfauntsch, K.M. Prise, B.C. Micheal, Radiat. Res. 156 (2001) 796.
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Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Biological studies using mammalian cell lines and the current status of the microbeam irradiation system, SPICE T. Konishi a,*, T. Ishikawa a, H. Iso a,b, N. Yasuda a, M. Oikawa a, Y. Higuchi a,b, T. Kato a,c, K. Hafer d, K. Kodama a,b, T. Hamano a, N. Suya a, H. Imaseki a a
Dept. of Technical Support and Development, Fundamental Technology Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Neos-Tech Co. Ltd., Benten 4-11-13-202, Chuo-ku, Chiba 206-0045, Japan c Graduate School of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshimaku, Tokyo 171-8501, Japan d Department of Radiation Oncology, UCLA School of Medicine, Los Angeles, CA, USA b
a r t i c l e
i n f o
Available online 13 March 2009 PACS: 29.27.Eg 29.27. a 61.80. x 87.50.Gi 87.80. y Keywords: SPICE Microbeam Protons Mammalian cells CR-39 Biological effect
a b s t r a c t The development of SPICE (single-particle irradiation system to cell), a microbeam irradiation system, has been completed at the National Institute of Radiological Sciences (NIRS). The beam size has been improved to approximately 5 lm in diameter, and the cell targeting system can irradiate up to 400– 500 cells per minute. Two cell dishes have been specially designed: one a Si3N4 plate (2.5 mm 2.5 mm area with 1 lm thickness) supported by a 7.5 mm 7.5 mm frame of 200 lm thickness, and the other a Mylar film stretched by pressing with a metal ring. Both dish types may be placed on a voice coil stage equipped on the cell targeting system, which includes a fluorescent microscope and a CCD camera for capturing cell images. This microscope system captures images of dyed cell nuclei, computes the location coordinates of individual cells, and synchronizes this with the voice coil motor stage and single-particle irradiation system consisting of a scintillation counter and a beam deflector. Irradiation of selected cells with a programmable number of protons is now automatable. We employed the simultaneous detection method for visualizing the position of mammalian cells and proton traversal through CR-39 to determine whether the targeted cells are actually irradiated. An immuno-assay was also performed against c-H2AX, to confirm the induction of DNA double-strand breaks in the target cells. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Single-cell microbeam irradiation systems have recently become important tools for radiation biology. The major characteristic of microbeam irradiation systems is a very narrow beam of radiation, micrometer size or smaller, corresponding to cellular or sub-cellular dimensions. In addition, microbeam irradiation of exactly one to any preset number of particles per cell allows study of radiation risks from very low doses to be addressed. Moreover, microbeam techniques can be used to address such questions as the effects of irradiation on unirradiated neighboring cells, such as the bystander effects, and the relative sensitivities of different parts of the cell. Multiple microbeam facilities have been developed for biological research [1–13]. In Japan an array of microbeam facilities with different radiation sources is available for biological studies [14–16]. Such facilities have been used to study low-dose exposure, hyper radio-sensitivity, bystander effects and others [17–21]. * Corresponding author. Tel.: +81 43 206 3032; fax: +81 43 206 3514. E-mail address: [email protected] (T. Konishi). 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.03.060
The single-particle irradiation system to cell, SPICE, at the National Institute of Radiological Sciences (NIRS) generates 3.4 MeV protons with an approximately 5 lm diameter beam, and is the only proton microbeam irradiation system in Japan [16]. SPICE is currently operational for biological studies. This paper describes the results of improvements of the beam line, along with preliminary data from biological experimentation. 2. Materials and methods 2.1. Outline of the microbeam irradiation system, SPICE The details of SPICE have already been described [16]. Briefly, the electrostatic accelerator facility of NIRS supplies protons and helium ions by a Tandetron accelerator (HVEE, High Voltage Engineering Europe Ltd.) [22]. Three horizontal beam lines are available for PIXE analysis. PIXE Analysis System and Tandetron Accelerator, PASTA, consists of a conventional PIXE line for analysis under vacuum conditions, an ‘‘in-air” PIXE line [23], and a microbeam scanning PIXE line for two-dimensional mapping of multi-elemental distributions [24]. The fourth beam line, SPICE is a vertical beam
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line which diverges from the microbeam scanning PIXE (microPIXE) line and transported upward by a 90° bending magnet installed in the middle of the micro-PIXE beam line. This vertical beam is focused by a mono-bloc triplet quadrupole lens (Q lens; OM170, Oxford microbeams, Ltd.) to exclude such low-energy particle components by scattering seen in microbeams produced by the collimation method [1]. 3.4 MeV protons extracted into atmosphere are used to irradiate cells. Because the beam diameter deteriorates outside of the vacuum system due to proton scattering in air, the distance the beam travels through air must be minimized. In our previous paper, we used Kapton film as a beam exit window. This film bends more than 300 lm toward the vacuum and this air gap prevents the beam from being focused to less than 10 lm in diameter. In this work, a 1 lm thick Si3N4 membrane (Silcon Ltd.) of 2.5 mm 2.5 mm area with a 200 lm thick frame of 7.5 mm 7.5 mm area was glued onto the beam exit pipe. A schematic diagram of the beam exit window and the configuration of the surrounding components are shown in Fig. 1(A). The air gap between the Mylar film of the cell dish and the beam exit window was set to be below 100 lm to keep the beam as focused as possible prior to reaching the cell position. The bending of the Si3N4 membrane is approximately 40–50 lm. The air gap was determined by measuring the
difference in operating distance of a 20 objective lens from the focused position at the surface of beam exit window to the surface of the Mylar film of the cell dish. It was adjusted by inserting a metal thin foil of suitable thickness between the stage and the steel plate of the cell dish. For the routine irradiation of cells, the beam intensity can be controlled to be less than 1.0 105 protons per second by adjusting the objective slits installed in the horizontal beam line. The number of protons traveling through the cells was counted using a scintillation detector set above the cell dish, as shown in Fig. 1. A fast beam deflector system (10 MHz) was installed upstream of the 90° bending magnet and was used to switch the beam off on demand. It consists of two parallel metal electrodes connected to a high-voltage generator. After the scintillation detector detects the preset number of protons that traversed through the CR-39 (or the cells), the computer that controls the voice coil motor stage gives a high-speed trigger pulse to the beam deflector which drops the voltage of one of the parallel electrodes to keep the beam from being transported into the vertical beam line. The voice coil motor stage is self-developed with a Technohands, Co., Ltd. (Yokohama, Japan), and has 10 mm 10 mm traveling range with a position accuracy of 40 nm. By repeating this procedure, it is possible to irradiate a given cell with a programmable number of protons. 2.2. Cell dish and microscope Two types of cell dishes were specially designed. Type one, previously described in [16], is a Si3N4 plate (7.5 7.5 mm frame) attached to a metal dish with Vaseline (Wako, Osaka). The cells are cultured on a 2.5 mm 2.5 mm area of 1 lm thickness, which minimizes the energy loss of protons before they reach the cells. A second type of dish was designed for two reasons: firstly, to culture a larger number of cells and secondly to reduce the air gap between the Mylar film to which the cells are attached and the beam exit window. Cells may be cultured on a 30 mm diameter area of a Mylar film stretched across a 30 mm diameter steel ring to 33 mm diameter hole, with the film in between. A photograph of this cell dish is shown in Fig. 1(B). It is placed on the voice coil stage of the cell targeting system, which also contains a fluorescent microscope (BX51, Olympus) and a CCD camera (ORCA-ER, Hamamatsu). An example of a fluorescent image of the cell nuclei is shown in Fig. 2, which displays the fluorescence of CHO-K1 cells that were dyed with
Fig. 1. Panel A is a schematic diagram of the end station of SPICE, and B is a photograph of the Mylar film cell dish.
Fig. 2. Fluorescent microscope image of CHO-K1 cells dyed with Hoechst 33258. Each cell nuclei is given a reference number for identification in subsequent biological experiments.
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Hoechst 33258 (Dojindo Laboratories, Kumamoto). Green ellipse shows results of automated cell recognition, which is a selfdeveloped algorithm based on a least-square technique for a second order poly-nominal function [25]. With this recognition algorithm, the X–Y coordinates of individual cells are obtained from the fluorescence signal of each nucleus. Each nucleus was given a number (shown in green) to identify the cells later in biological experiments. Therefore, as according to the sample-preparation protocol, cells were cultured in media containing 1 lM Hoechst 33258 for 2–3 h to stain the nuclei sufficiently for cell recognition from the fluorescent image. The voice coil motor stage is synchronized with a single-particle irradiation system described in the previous section. 2.3. Beam-size measurement using CR-39 The actual beam size was determined by irradiating a 20 mm diameter size and 110 lm thick plastic track detector, CR-39 (HARZLAS TD-1, Fukuvi chemical industry), adhered to the Mylar surface of a cell dish. Fig. 3 shows the irradiated CR-39, etched in 7 M NaOH at 70° for 2 h prior to capturing the image with a confocal laser microscope (Fluoview1000, Olympus). Fig. 3(A) shows the logo mark of the NIRS on the CR-39. Each position was irradiated with 10 protons for a total of 2704 positions with a 10 lm pitch. Panel B shows an expanded image of the area, B, shown in panel A. The irradiation was performed automatically according to an input text file that contains the desired preset number of protons and the X–Y coordinates of the target positions. This system enables one to irradiate 6–8 positions per second, thus the irradiation of the CR-39 shown
Fig. 3. Panel A shows the logo of the National Institute of Radiological Sciences, NIRS on the CR-39. Each position was irradiated with 10 protons for a total of 2704 positions at 10 lm pitch. Bar size, 100 lm. Panel B shows an expanded image of the area, B, shown in panel A. Bar size, 20 lm. Panel C shows the beam size of 20, 50, 100, 150, 200 protons.
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in Fig. 3(A) was completed in less than 10 min. Panel C shows a CR-39 irradiated with 20, 50, 100, 150, 200 protons per position with a 50 lm pitch. The beam size for 200 protons was approximately 5 lm in diameter.
3. Preliminary biological results 3.1. Simultaneous detection of the beam and a cell nucleus We employed a contact microscopy technique [26], previously developed for imaging boron in boron neutron capture therapy to determine whether the targeted HeLa cells (human cervix carcinoma cell line, purchased from Japanese Collection of Research Bioresources Cell Bank, JCRB) are actually irradiated by SPICE. The method is based on the fact that etching speed of CR-39 is enhanced by UV exposure [27]. It is applied to a contact UV microscopy technique proposed by Amemiya et al., in which a relief image of a slice of rat tissue attached to the CR-39 surface and etch pits by neutron-induced alpha-particles were simultaneously recorded on the CR-39 surface [26,28]. This method enables visualization of mammalian cells as a relief on a CR-39 simultaneously with the etch pits that indicate the positions of ion traversals. This technique is very convenient, and enables one to simultaneously obtain geometric information of the cells and ion traversals using only common laboratory equipment, such as a conventional optical microscope, a UV lamp, and commercially available CR-39. HeLa cells were cultured on the surface of CR-39 approximately 24 h before irradiation. Culture conditions were described previously [16,29]. Cells were stained with 1 lM Hoechst 33258 before being irradiated to determine the cell positions in the dish (see Fig. 4(C)). The 3.4 MeV protons traversed the 100 lm air gap, 110 lm of CR-39, the cell nucleus, and finally reached the scintillation counter. After irradiation, the cells were fixed with ice-cooled methanol:acetone = 1/1 solution for 10 min, and rinsed with distilled water. They were then dried for a day in air. The CR-39 plate was exposed to a total dose of 1.6 105 J/m2 of a UV light (mainly 254 nm) with a low-pressure mercury lamp (GL-10, Toshiba) for 4 h. The cells were removed from the CR-39 film, which was then etched in 7 M NaOH for 2 h to detect the etch pits and the embossment of the cell relief. Fig. 4(A) shows an image of HeLa cells as an embossment on the CR-39 surface. Panel A shows an embossment on the CR-39 of the non-targeted cells and the targeted cells, which were irradiated with 500 protons. The targeted cells are indicated by the arrows in panel A, and panel B is a magnified image of B in panel A. The etch pits produced by protons can be clearly identified as well as the cell nuclei on CR-39, showing that targeted cells were actually traversed by the protons. Panel C shows the fluorescence of the cell nuclei. The beam size (etch pits) seen in Fig. 4, is the size of outgoing beam from the 110 lm thick CR-39. The beam size of entering side of CR-39 was approximately 3–4 time smaller in diameter compared to those seen on the outgoing size. This is because of the proton scatter during traversal through the CR-39. Although the etch pits were not detected in the area surrounding the cell nuclei, this was not due to the etching process. If protons traversed somewhere outside of the cell area, larger size etch pits than those in the cell relief should be identified because of the enhanced etching velocity of CR-39 by the previous UV exposure [25–27]. Therefore, we estimated that the beam was targeted specifically to the cell nucleus, although we cannot exclude the possibility that the whole cell area was irradiated. Thus further statistical measurements for beam targeting accuracy are necessary.
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Fig. 4. (A) Microscope overview image of HeLa cell positions as an embossment on the CR-39 surface. (B) In enlarged view of the area indicated in A, where the irradiated cells are indicated by arrows. (C) Displays the fluorescence image captured from area C. Bar size, 50 lm.
Fig. 5. Panel A represents an image of cell nuclei stained by Hoechst 33258, and panel B is a fluorescent image of c-H2AX. Those number shown in green in panel A is the identification number for the individual cell nucleus. Panels A and B are merged in panel C, showing the nuclei in violet, and c-H2AX in green. The number of protons irradiated to targeted cell nucleus is indicated by the number shown in panel C. Bar size, 20 lm. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)
3.2. Immuno-assay for the detection of induced DNA double-strand breaks in targeted cell nuclei The purpose of this preliminary experiment was to determine whether DNA double-strand breaks (DSBs) were induced in the targeted cell. We visualized the DSBs induced by the traversals of protons through the cell nucleus using phosphorlylated histone,
H2AX (c-H2AX), as a marker for DNA DSBs [29,30]. CHO-K1 cells were prepared and cultured on the cell dish 24 h before irradiation. Approximately, 2 105 cells were inoculated on the Mylar film. After staining the cells with Hoechst 33258, the cell dish was set on the voice coil motor stage for image capturing and cell recognition. Targeted cells were selected according to the numbers given in the image (Fig. 5(A)). The number of protons to be irradiated
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to those targeted cells was preset, as shown in Fig. 5. After irradiation, the cells were incubated at 37° with 5% CO2/95% air for 30 min for the phosphorylation of H2AX to reach its maximum and then the cells were fixed with 2 ml of 4% paraformaldehyde for 15 min at room temperature. An immuno-fluorescent assay was held using the anti-c-H2AX antibody (CHEMICON International, Inc) and secondary anti-rabbit Alexa 488 antibodies (Molecular Probes), as mentioned elsewhere [16,28]. Fig. 5(B) shows the fluorescence of immuno-stained c-H2AX. Fig. 5(C) is a merged view of panel A and B. The fluorescence of c-H2AX was identified in all the targeted cells, and not in the non-targeted cell nuclei. In addition, the fluorescence increased corresponding to the number of protons irradiated. As a result, the image obtained by UV imaging technique (Fig. 5) and the immuno-stained image of cH2AX (Fig. 5) are the evidences that the cells were targeted accurately. 4. Conclusion Improvements on the microbeam irradiation system, SPICE, have been made. Approximately, 5 lm in beam diameter can be achieved. For cell irradiation, the X–Y coordinates of cells in a dish can be calculated according to the fluorescent cell nuclei imaged by fluorescent microscope. The irradiation procedures can be automatically performed and the maximum speed for cell irradiation is 400–500 cells per minute. The targeting accuracy was checked by employing the simultaneous detection method, which visualized HeLa cells as embossment reliefs and beam position as etch pits on the surface of CR-39. Also CHO-K1 cells were irradiated and immuno-stained against c-H2AX to see an induction of DNA double-strand breaks in the targeted cells. Both experiments showed that SPICE is operational for biological experiments. Further development is underway in order to increase the performance of the microbeam irradiation system to perform studies targeting cell nuclei. References [1] M. Folkard, B. Vojnovic, K.M. Prise, A.G. Bowey, R.J. Locke, G. Schettino, B.D. Micheal, Int. Radiat. Biol. 72 (1997) 375. [2] G. Randers-Pehrson, C.R. Geard, G. Johnson, C.D. Elliston, D.J. Brenner, Radiat. Res. 156 (2001) 210. [3] M. Folkard, G. Schettino, B. Vojnovic, S. Gilchrist, A.G. Michette, S.J. Pfauntsch, K.M. Prise, B.C. Micheal, Radiat. Res. 156 (2001) 796.
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