- Email: [email protected]
Microirradiation of cells with energetic heavy ions
Nuclear Instruments and Methods in Physics Research B 231 (2005) 195–201 www.elsevier.com/locate/nimb
Microirradiation of cells with energetic heavy ions G. Dollinger a,*, V. Hable a, A. Hauptner b, R. Kru¨cken b, P. Reichart b, A.A. Friedl c, G. Drexler c, T. Cremer d, S. Dietzel d a LRT 2, Universita¨t der Bundeswehr Mu¨nchen, D-85579 Neubiberg, Germany Physik-Department E12, Technische Universita¨t Mu¨nchen, D-85748 Garching, Germany c Strahlenbiologisches Institut, Ludwigs-Maximilians Universita¨t Mu¨nchen, D-80336 Mu¨nchen, Germany Department Biologie II, Ludwigs Maximilians Universita¨t Mu¨nchen, Richart Wagner Str. 10/I, D-80333 Mu¨nchen, Germany b
d
Available online 7 April 2005
Abstract The ion microprobe SNAKE (superconducting nanoscope for applied nuclear (Kern) physics experiments) at the Munich 14 MV tandem accelerator achieves beam focusing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. This allows to adjust the number of DNA single strand breaks (SSBs) and double strand breaks (DSBs) per ion and per cell nucleus from about 0.1 DSBs per ion to several 100 DSBs per ion. When irradiating with single 100 MeV 16O ions, the adapted setup permits a fwhm irradiation accuracy of 0.55 lm in x-direction and 0.4 lm in y-direction, as demonstrated by retrospective track etching of polycarbonate foils. The experiments point to investigate protein dynamics after targeted irradiation. As an example for such experiments we show a kind of three dimensional representation of foci of c–H2AX which are visible 0.5 h after the irradiation with 100 MeV 16O ions took place. It shows the gross correlation with the irradiation pattern but also distinct deviations which are attributed to protein dynamics in the cell. 2005 Elsevier B.V. All rights reserved.
1. Introduction Focused ion beams are useful tools to study the influence of ionising radiation on living cells. An even wider field opens when the local ion–cell * Corresponding author. Tel.: +49 89 6004 3505; fax: +49 89 6004 3295. E-mail address: [email protected] (G. Dollinger).
interaction is used to introduce quantifiable damage at a certain point for a given time and to investigate the induced dynamics of signal processes and repair proteins, e.g. to study cell surveillance strategies. The nice characteristic of ion irradiation is that damage from ionisation, activation or nuclear–nuclear knock on reactions is localised in a limited region around the ion track. If a certain place is targeted within a cell the following reactions in the cell can be studied in the three spatial
0168-583X/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2005.01.056
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Fig. 1. Principle of single cell irradiation. (a) HeLa cells visualised by optical phase contrast microscopy at the focal plane of SNAKE are targeted by the ion beam focus. A counted number of ions (i.e. one ion) are shot in a certain positional arrangement. (b) After irradiation the induced reactions are analysed at the irradiation site and in the surroundings. Characteristics of one cell seen in the phase contrast microscopy are schematically sketched.
dimensions and in dependence on time as it is illustrated in Fig. 1. The figure shows a phase contrast micrograph of HeLa cells as it was obtained from our optical microscope at the focal plane of the Munich ion microprobe SNAKE (superconducting nanoscope for applied nuclear (Kern) physics experiments) [1]. If one certain place is targeted by a single ion (Fig. 1(a)) the local biological-chemical dynamics stimulated by the damage along the ion track as well as in the surrounding nucleus or even the surrounding cells can be investigated. The last mentioned approach has already been applied intensely using micro-collimated ion beams to investigate the famous bystander effect [2,3]. In this article we summarise the adaption of SNAKE to perform irradiation of biological cells under living conditions. Especially, we discuss the wide ion and energy spectrum available at SNAKE and why it gives new perspectives for the application of focused ion beams compared to most other actual ion microprobes. In addition we show one experiment where we demonstrate how protein dynamics and arrangement in the cell nucleus can be followed.
2. Damage potential of single ion irradiation SNAKE has, compared to most other ion microprobes, which are used for biological studies, the widest ion spectrum available. This is because a 14 MV tandem accelerator is used at SNAKE
which delivers enough energy and therewith a range of at least 25 lm in water for all relevant ions. Such a large range is necessary to transmit ions from vacuum through thin foils into cells at living conditions and through the cells themselves as well as to deposit enough energy in an ion detector. There are protons and helium beams as well as any other heavy ion beam available with the exception of the heavier noble gas elements which cannot be accelerated by a tandem accelerator, as used at SNAKE. The only heavy ion microprobe which is used for experiments in cell biology up to now is the one at GSI [4]. However, this ion probe will only be able to handle ions heavier than carbon due to the single ion detection system used. Thus, systematic studies from loosly ionising radiation to strong ionisation also cannot be performed at this ion microprobe. The only probe with submicrometer resolution, which has light ions as well as heavy ion beams available for biological experiments seems to be SNAKE at the moment. The main issue with respect to cell irradiation by single ions is the number of damaged molecules introduced by a single ion and, in a second stage, their density distribution around the ion track. In the following we will give a rough estimate of the number of DNA single strand breaks (SSBs) and double strand breaks (DSBs) produced by a single ion in one cell nucleus with respect to the nuclear charge and the energy of the ion. In Fig. 2 we have plotted the electronic stopping forces (linear energy transfer (LET) value) versus velocity
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Fig. 2. Damage potential of various ions in dependence of their energy. The electronic stopping force (LET, right abscissa) and the energy deposited in a fictitious cell (left abscissa) are plotted versus ion velocity (bottom ordinate) and specific ion energy (top ordinate). According to the volume of the HeLa cell nucleus of about 715 lm3 an average dose can be calculated (second right abscissa). Assuming 1000 single strand breaks (SSBs) per Gray and 35 double strand breaks (DSBÕs) per Gray the number of SSBs and DSBs can be calculated in this linear approximation (third and fourth right abscissa). For the high dose regime this linear relationship does not hold and the calculated numbers may be only a lower estimate in general (shaded area).
(specific ion energy) for a selection of ions from protons to gold ions according to the SRIM 2003 code [5]. The ion energies which are available at SNAKE are marked by solid lines while these regions are extended by dotted lines. Nuclear energy loss is less than 1% in the plotted energy range and is neglected in the following discussion although there might be significant contribution to cell damage due to the different quality of damage which is introduced from atoms kicked off from their sites. Assuming an average height of a cell nucleus (in our case 7.15 lm for a HeLa cell grown on Mylar foil [6]) one can calculate the total energy deposited in the cell nucleus as it is shown on the left abscissa in Fig. 2. From that value and an average volume (mass) of the cell nucleus of 715 lm3 (715 pg) [6] one get the dose in the cell nucleus as read from the first separated right abscissa in Fig. 2. With the value of 1000 SSBs and 35 DSBs per Gray for sparsely ionising irradiation [7,8] we can extract an abscissa of SSBs and DSBs induced per ion in a single cell nucleus (see the two right abscissas in Fig. 2). The linear extrapolation of the SSBs and DSBs is an approximation: at lar-
ger ionisation densities the number of DSBs increases due to the overlapping of ionisation regions of the secondary electrons. This effect is normally taken into consideration introducing a relative biological effectiveness (RBE) factor for DSB induction larger than 1. The actual RBE factors for SSBs and DSBs in dependence of the ions nuclear charge and its energy are, however, not known. Therefore, the corresponding area has been shaded on the DSB-abscissa. As a rough estimate, the linearly extrapolated values will be taken for DSB-induction keeping in mind that it is only a lower limit for the densely ionising radiation. For the experiments we describe here we have used a 100 MeV oxygen beam which produces 23 DSBs per ion and per cell nucleus according to the described linear approximation. Fig. 2 shows, that this number of DSBs per ion can be varied over more than 3 orders of magnitude, from loosly ionising irradiation of proton beams at 20 MeV with in average only 1/10 DSB per proton to high density ionisation of more than 200 DSBs per ion by choosing the appropriate ion and ion energy. This manifests the attraction to perform cell
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biological studies at SNAKE. Moreover, the high energies available at SNAKE ensure sufficient ranges to make cell irradiation possible even when heavy ions are used for the irradiation. In addition angular scattering can be kept small enough to keep overall positional resolution.
3. Experimental The preparation of single ions at SNAKE has already been discussed in a previous paper [9]. Here we will give a short overview on the main features and show actual values of the performance for single cell irradiation. The ions are normally produced in a high current cesium ion sputter source. Only the light gaseous ions are produced using a duoplasmatron source or a newly installed ECR ion source using a cesium vapour charge exchange section to obtain a negatively charged beam for the injection into the Munich tandem accelerator [10]. The beam is cut downstream from a 90 analysing magnet by specially designed microslits [11] to achieve a beam size of about 16 · 8 lm2. The divergence of the beam is cut by an additional pair of slits 8 m downstream. A typical aperture of 50 · 50 lm2 is used to cut the beam close to 1 kHz. The normalised brilliance of the oxygen beam in front of the superconducting quadrupole lense has been measured to be 1 pnA/(mm2 mrad2 MeV) which is even sufficient for high current applications at larger divergence apertures. The focal plane used for cell irradiation is positioned about 20 cm downstream from the optimal focus of the original SNAKE configuration in order to get optimal conditions for cell handling. This position reduces the demagnification factors to 1/88 in x-direction and 1/24 in y-direction. Higher demagnification factors are obtained using a two-stage demagnification adding an additional quadrupole 24 m in front of the superconducting lense. However, this two-stage option has not been applied up to now for cell irradiation. The ions are transmitted through an exit window of 7.5 lm Kapton which is mounted on a movable exit nozzle of 1 mm diameter (Fig. 3). The beam is focused utilising a CsI crystal already situated at the irradiation plane in air. The optical
Fig. 3. Schematical arrangement at the focal plane of SNAKE for living cell irradiation. Ions are transmitted into air through an 7.5 lm thick Kapton foil. Cells are placed between two 6 lm thick Mylar foils in a saturated atmosphere in order to prevent drying of the cells and positioned in close contact to the exit window. This configuration allows cell survival for more than 0.5 h. Within such a period the cells are targeted by an optical phase contrast microscope (a) and irradiated (b). The detector is mounted on the turret of the optical microscope and is quickly replaced by the objective lense or vice versa.
microscope mounted behind the sample position (Fig. 3(a)) allows a focusing of the ion beam below 1 lm diameter. The luminosity of the CsI and the aperture of the optical microscope is sufficient to obtain an image of the beam with a beam current as low as 200 oxygen ions per second. The beam is focused by adjusting the currents in the three quadrupole sections of the superconducting lens (the system is magnetooptically a doublet system because the last two sections have the same polarity), a correction of spherical aberration by adjusting the hexapole components by additional correction loops installed on the quadrupoles (but spherical aberration has no influence on the small emittance beams used in the biological experiments) and by adjusting a homogeneous 50 Hz sinusoidal magnetic field in front of the lens in its phase and amplitude in order to compensate
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Fig. 4. The pointing resolution of the 100 MeV oxygen beam is examined from a regular matrix which has been shot onto a polycarbonate foil which is etched and examined by an optical microscope. Fitting a regular grid to the matrix the distribution of distances of these points from the grid are plotted. The standard deviation of these distributions is in the range of ±0.5 lm in both xand y-direction.
for 50 Hz beam fluctuations induced by external stray fields. This stray field causes a fluctuating beam spot with up to 2 lm amplitude in the focal plane without correction. It is therefore necessary to compensate this 50 Hz oscillation in order to get an effective beam spot below 1 lm. It has been proven that compensation works well with the same setting for several weeks which enables stable irradiation conditions for long terms. Single ions are produced using a fast electrostatic beam deflector about 5 m in front of the divergence slits. It deflects the beam until an ion has to be delivered. Then the deflector is switched off until one ion has met the focal plane and has been transmitted to an ion detector which is put behind the cell instead of the microscope objective (Fig. 3(b)). The beam is stopped within 1.4 ls reaction time after ion detection. This reaction time is dominated by the traveling time of an ion from the deflector to the focal plane. The quality of the beam at the irradiation position in air has been measured by analysing a polycarbonate foil as a nuclear track detector which has been irradiated by a point matrix with a pitch
of 5 lm · 5 lm under similar conditions as the cells later on. After etching the foil in alcoholic base one sees each ion hit in the foil as a dark point under bright field illumination in the optical microscope (Fig. 4(a)). Assuming a regular grid, as marked in red in Fig. 4(a), the distribution of distances of the points from the grid can be plotted in x- and y-direction independently. These distributions are shown in Fig. 4(b) and (c). A full width half maximum of 0.55 lm in x-direction and 0.4 lm in ydirection is evaluated which is some factors better than reported earlier [9]. It is close to the resolution of conventional optical microscopy and is therefore in line with the requirements to study protein dynamics in cells by optical microscopy after targeting a certain place in the single cell.
4. Matrix irradiation and repair foci in cell nuclei Up to now, most of our biological experiments have been performed by irradiating regular patterns onto a field of 500 · 500 lm2 where cells are densely grown onto a 6 lm thick Mylar foil. Such a regular
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Fig. 5. Data of Immunofluorescence images of p53BP1 binding protein1 (p53BP1)s accumulation in a single HeLa cell nucleus irradiated by matrix irradiation (5 · 5 lm pitch). Raw images are acquired by three dimensional microscopy, the fluorescence images are processed using the deconvolution algorithm Huygens [12]. The images represent the 53BP1 accumulation under the view of four differenct angles. Although the regular structure of the matrix can be recognised, the whole structure is slightly deformed as well as the line structure of each single ion track.
pattern can be the 5 lm · 5 lm point matrix as it was used for the evaluation of the beam resolution. Due to the possibility of a fast scanning and recognition (up to 100 ions per second at the moment, 1000 ions per second possible in future) such patterns are written within a few minutes which is not possible with collimated ion beams. Several experiments deal with the observation of the accumulation of repair proteins in foci along the ion track by immuno-fluorescence techniques on those irradiated cells after a certain time of further cell growth. Cell growth conditions and cell treatment after irradiation is described in detail in [9]. Micrographs of the fluorescence signals from secondary antibodies marking c–H2AX (the phosphorilated histon H2AX) in a single cell nucleus are shown in Fig. 5. The figure shows four different angular views of one three dimensional data set from optical microscopy of a cell which has been fixed 1.5 h after irradiation. The individual line spots show the c–H2AX mainly arranged along
the 5 · 5 lm2 point matrix shot with single 100 MeV oxygen ions and show only minor deviation from the gross irradiation pattern. However, the lines are not really straight lines but show curvatures and a bulky structure which are not expected from angular deflections of the ions. The line in the middle is rather faded while the lines around show more or less a continuous structure. The different behaviour of line spots inside the cell nucleus and at the side as well as the line structure behaviour are commonly recognised and change with the time between irradiation and fixation. These data are subjected to an ongoing analysis in order to get a detailed knowledge on protein dynamics and cell repair.
5. Conclusion It has been demonstrated that the availability of a wide ion spectrum and high ion energies at a
G. Dollinger et al. / Nucl. Instr. and Meth. in Phys. Res. B 231 (2005) 195–201
focused ion microprobe allow a new quality of experiments in radio-biology and in cell-biology in general. Fast scanning makes it possible to apply a certain irradiation pattern to the cells with many single shots. Heavy ions make enough damage that the track of a single ion is sufficient to see localised protein expression in the cell. It offers the opportunity to study repair dynamics in cells in three space coordinates beginning with well defined starting points in space and time.
Acknowledgement This work is supported by the Maier-LeibnitzLaboratorium der LMU und TU Mu¨nchen.
References [1] G. Dollinger, G. Datzmann, A. Hauptner, R. Hertenberger, H.-J. Ko¨rner, P. Reichart, B. Volkaerts, Nucl. Instr. and Meth. B 210 (2003) 6, and references herein.
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[2] D.J. Brenner, E.J. Hall, Radiat. Prot. Dosim. 99 (2002) 283. [3] K.M. Prise, M. Folkard, B.D. Micheal, Radiat. Prot. Dosim. 104 (2003) 347. [4] B.E. Fischer, M. Heiß, M. Cholewa, Nucl. Instr. and Meth. B 210 (2003) 285. [5] J.F. Ziegler, J.P. Biersack, U. Littmark, The Stopping and Range of Ions in Matter, Pergamon Press, New York, 1985. [6] L. Tartier, C. Spenlehauer, H.C. Newman, M. Folkard, K.M. Prise, B.D. Micheal, J. Menissier-de Murcia, G. de Murcia, Mutagenesis 18 (2003) 411. [7] A.A. Friedl, A. Kraxenberger, F. Eckardt-Schupp, Electrophoresis 16 (1995) 1865. [8] F. Kraxenberger, K.J. Weber, A.A. Friedl, F. EckardtSchupp, M. Flentje, P. Quicken, A.M. Kellerer, Radiat. Environ. Biophys. 37 (1998) 107. [9] A. Hauptner, S. Dietzel, G.A. Drexler, P. Reichart, R. Kru¨cken, T. Cremer, A.A. Friedl, G. Dollinger, Radiat. Environ. Biophys. 42 (2004) 237. [10] R. Hertenberger, Y. Eisermann, A. Hofmann, A. Metz, P. Schiemenz, S. Trieb, G. Graw, Rev. Sci. Instr. 69 (1998) 750. [11] O. Schmelmer, G. Dollinger, G. Datzmann, C. Goeden, H.-J. Ko¨rner, Nucl. Instr. and Meth. B 158 (1999) 107. [12] Scientific Volume Imaging b.v., URL http://www.svi.n1/.
Microirradiation of cells with energetic heavy ions G. Dollinger a,*, V. Hable a, A. Hauptner b, R. Kru¨cken b, P. Reichart b, A.A. Friedl c, G. Drexler c, T. Cremer d, S. Dietzel d a LRT 2, Universita¨t der Bundeswehr Mu¨nchen, D-85579 Neubiberg, Germany Physik-Department E12, Technische Universita¨t Mu¨nchen, D-85748 Garching, Germany c Strahlenbiologisches Institut, Ludwigs-Maximilians Universita¨t Mu¨nchen, D-80336 Mu¨nchen, Germany Department Biologie II, Ludwigs Maximilians Universita¨t Mu¨nchen, Richart Wagner Str. 10/I, D-80333 Mu¨nchen, Germany b
d
Available online 7 April 2005
Abstract The ion microprobe SNAKE (superconducting nanoscope for applied nuclear (Kern) physics experiments) at the Munich 14 MV tandem accelerator achieves beam focusing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. This allows to adjust the number of DNA single strand breaks (SSBs) and double strand breaks (DSBs) per ion and per cell nucleus from about 0.1 DSBs per ion to several 100 DSBs per ion. When irradiating with single 100 MeV 16O ions, the adapted setup permits a fwhm irradiation accuracy of 0.55 lm in x-direction and 0.4 lm in y-direction, as demonstrated by retrospective track etching of polycarbonate foils. The experiments point to investigate protein dynamics after targeted irradiation. As an example for such experiments we show a kind of three dimensional representation of foci of c–H2AX which are visible 0.5 h after the irradiation with 100 MeV 16O ions took place. It shows the gross correlation with the irradiation pattern but also distinct deviations which are attributed to protein dynamics in the cell. 2005 Elsevier B.V. All rights reserved.
1. Introduction Focused ion beams are useful tools to study the influence of ionising radiation on living cells. An even wider field opens when the local ion–cell * Corresponding author. Tel.: +49 89 6004 3505; fax: +49 89 6004 3295. E-mail address: [email protected] (G. Dollinger).
interaction is used to introduce quantifiable damage at a certain point for a given time and to investigate the induced dynamics of signal processes and repair proteins, e.g. to study cell surveillance strategies. The nice characteristic of ion irradiation is that damage from ionisation, activation or nuclear–nuclear knock on reactions is localised in a limited region around the ion track. If a certain place is targeted within a cell the following reactions in the cell can be studied in the three spatial
0168-583X/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2005.01.056
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G. Dollinger et al. / Nucl. Instr. and Meth. in Phys. Res. B 231 (2005) 195–201
Fig. 1. Principle of single cell irradiation. (a) HeLa cells visualised by optical phase contrast microscopy at the focal plane of SNAKE are targeted by the ion beam focus. A counted number of ions (i.e. one ion) are shot in a certain positional arrangement. (b) After irradiation the induced reactions are analysed at the irradiation site and in the surroundings. Characteristics of one cell seen in the phase contrast microscopy are schematically sketched.
dimensions and in dependence on time as it is illustrated in Fig. 1. The figure shows a phase contrast micrograph of HeLa cells as it was obtained from our optical microscope at the focal plane of the Munich ion microprobe SNAKE (superconducting nanoscope for applied nuclear (Kern) physics experiments) [1]. If one certain place is targeted by a single ion (Fig. 1(a)) the local biological-chemical dynamics stimulated by the damage along the ion track as well as in the surrounding nucleus or even the surrounding cells can be investigated. The last mentioned approach has already been applied intensely using micro-collimated ion beams to investigate the famous bystander effect [2,3]. In this article we summarise the adaption of SNAKE to perform irradiation of biological cells under living conditions. Especially, we discuss the wide ion and energy spectrum available at SNAKE and why it gives new perspectives for the application of focused ion beams compared to most other actual ion microprobes. In addition we show one experiment where we demonstrate how protein dynamics and arrangement in the cell nucleus can be followed.
2. Damage potential of single ion irradiation SNAKE has, compared to most other ion microprobes, which are used for biological studies, the widest ion spectrum available. This is because a 14 MV tandem accelerator is used at SNAKE
which delivers enough energy and therewith a range of at least 25 lm in water for all relevant ions. Such a large range is necessary to transmit ions from vacuum through thin foils into cells at living conditions and through the cells themselves as well as to deposit enough energy in an ion detector. There are protons and helium beams as well as any other heavy ion beam available with the exception of the heavier noble gas elements which cannot be accelerated by a tandem accelerator, as used at SNAKE. The only heavy ion microprobe which is used for experiments in cell biology up to now is the one at GSI [4]. However, this ion probe will only be able to handle ions heavier than carbon due to the single ion detection system used. Thus, systematic studies from loosly ionising radiation to strong ionisation also cannot be performed at this ion microprobe. The only probe with submicrometer resolution, which has light ions as well as heavy ion beams available for biological experiments seems to be SNAKE at the moment. The main issue with respect to cell irradiation by single ions is the number of damaged molecules introduced by a single ion and, in a second stage, their density distribution around the ion track. In the following we will give a rough estimate of the number of DNA single strand breaks (SSBs) and double strand breaks (DSBs) produced by a single ion in one cell nucleus with respect to the nuclear charge and the energy of the ion. In Fig. 2 we have plotted the electronic stopping forces (linear energy transfer (LET) value) versus velocity
G. Dollinger et al. / Nucl. Instr. and Meth. in Phys. Res. B 231 (2005) 195–201
197
Fig. 2. Damage potential of various ions in dependence of their energy. The electronic stopping force (LET, right abscissa) and the energy deposited in a fictitious cell (left abscissa) are plotted versus ion velocity (bottom ordinate) and specific ion energy (top ordinate). According to the volume of the HeLa cell nucleus of about 715 lm3 an average dose can be calculated (second right abscissa). Assuming 1000 single strand breaks (SSBs) per Gray and 35 double strand breaks (DSBÕs) per Gray the number of SSBs and DSBs can be calculated in this linear approximation (third and fourth right abscissa). For the high dose regime this linear relationship does not hold and the calculated numbers may be only a lower estimate in general (shaded area).
(specific ion energy) for a selection of ions from protons to gold ions according to the SRIM 2003 code [5]. The ion energies which are available at SNAKE are marked by solid lines while these regions are extended by dotted lines. Nuclear energy loss is less than 1% in the plotted energy range and is neglected in the following discussion although there might be significant contribution to cell damage due to the different quality of damage which is introduced from atoms kicked off from their sites. Assuming an average height of a cell nucleus (in our case 7.15 lm for a HeLa cell grown on Mylar foil [6]) one can calculate the total energy deposited in the cell nucleus as it is shown on the left abscissa in Fig. 2. From that value and an average volume (mass) of the cell nucleus of 715 lm3 (715 pg) [6] one get the dose in the cell nucleus as read from the first separated right abscissa in Fig. 2. With the value of 1000 SSBs and 35 DSBs per Gray for sparsely ionising irradiation [7,8] we can extract an abscissa of SSBs and DSBs induced per ion in a single cell nucleus (see the two right abscissas in Fig. 2). The linear extrapolation of the SSBs and DSBs is an approximation: at lar-
ger ionisation densities the number of DSBs increases due to the overlapping of ionisation regions of the secondary electrons. This effect is normally taken into consideration introducing a relative biological effectiveness (RBE) factor for DSB induction larger than 1. The actual RBE factors for SSBs and DSBs in dependence of the ions nuclear charge and its energy are, however, not known. Therefore, the corresponding area has been shaded on the DSB-abscissa. As a rough estimate, the linearly extrapolated values will be taken for DSB-induction keeping in mind that it is only a lower limit for the densely ionising radiation. For the experiments we describe here we have used a 100 MeV oxygen beam which produces 23 DSBs per ion and per cell nucleus according to the described linear approximation. Fig. 2 shows, that this number of DSBs per ion can be varied over more than 3 orders of magnitude, from loosly ionising irradiation of proton beams at 20 MeV with in average only 1/10 DSB per proton to high density ionisation of more than 200 DSBs per ion by choosing the appropriate ion and ion energy. This manifests the attraction to perform cell
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G. Dollinger et al. / Nucl. Instr. and Meth. in Phys. Res. B 231 (2005) 195–201
biological studies at SNAKE. Moreover, the high energies available at SNAKE ensure sufficient ranges to make cell irradiation possible even when heavy ions are used for the irradiation. In addition angular scattering can be kept small enough to keep overall positional resolution.
3. Experimental The preparation of single ions at SNAKE has already been discussed in a previous paper [9]. Here we will give a short overview on the main features and show actual values of the performance for single cell irradiation. The ions are normally produced in a high current cesium ion sputter source. Only the light gaseous ions are produced using a duoplasmatron source or a newly installed ECR ion source using a cesium vapour charge exchange section to obtain a negatively charged beam for the injection into the Munich tandem accelerator [10]. The beam is cut downstream from a 90 analysing magnet by specially designed microslits [11] to achieve a beam size of about 16 · 8 lm2. The divergence of the beam is cut by an additional pair of slits 8 m downstream. A typical aperture of 50 · 50 lm2 is used to cut the beam close to 1 kHz. The normalised brilliance of the oxygen beam in front of the superconducting quadrupole lense has been measured to be 1 pnA/(mm2 mrad2 MeV) which is even sufficient for high current applications at larger divergence apertures. The focal plane used for cell irradiation is positioned about 20 cm downstream from the optimal focus of the original SNAKE configuration in order to get optimal conditions for cell handling. This position reduces the demagnification factors to 1/88 in x-direction and 1/24 in y-direction. Higher demagnification factors are obtained using a two-stage demagnification adding an additional quadrupole 24 m in front of the superconducting lense. However, this two-stage option has not been applied up to now for cell irradiation. The ions are transmitted through an exit window of 7.5 lm Kapton which is mounted on a movable exit nozzle of 1 mm diameter (Fig. 3). The beam is focused utilising a CsI crystal already situated at the irradiation plane in air. The optical
Fig. 3. Schematical arrangement at the focal plane of SNAKE for living cell irradiation. Ions are transmitted into air through an 7.5 lm thick Kapton foil. Cells are placed between two 6 lm thick Mylar foils in a saturated atmosphere in order to prevent drying of the cells and positioned in close contact to the exit window. This configuration allows cell survival for more than 0.5 h. Within such a period the cells are targeted by an optical phase contrast microscope (a) and irradiated (b). The detector is mounted on the turret of the optical microscope and is quickly replaced by the objective lense or vice versa.
microscope mounted behind the sample position (Fig. 3(a)) allows a focusing of the ion beam below 1 lm diameter. The luminosity of the CsI and the aperture of the optical microscope is sufficient to obtain an image of the beam with a beam current as low as 200 oxygen ions per second. The beam is focused by adjusting the currents in the three quadrupole sections of the superconducting lens (the system is magnetooptically a doublet system because the last two sections have the same polarity), a correction of spherical aberration by adjusting the hexapole components by additional correction loops installed on the quadrupoles (but spherical aberration has no influence on the small emittance beams used in the biological experiments) and by adjusting a homogeneous 50 Hz sinusoidal magnetic field in front of the lens in its phase and amplitude in order to compensate
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Fig. 4. The pointing resolution of the 100 MeV oxygen beam is examined from a regular matrix which has been shot onto a polycarbonate foil which is etched and examined by an optical microscope. Fitting a regular grid to the matrix the distribution of distances of these points from the grid are plotted. The standard deviation of these distributions is in the range of ±0.5 lm in both xand y-direction.
for 50 Hz beam fluctuations induced by external stray fields. This stray field causes a fluctuating beam spot with up to 2 lm amplitude in the focal plane without correction. It is therefore necessary to compensate this 50 Hz oscillation in order to get an effective beam spot below 1 lm. It has been proven that compensation works well with the same setting for several weeks which enables stable irradiation conditions for long terms. Single ions are produced using a fast electrostatic beam deflector about 5 m in front of the divergence slits. It deflects the beam until an ion has to be delivered. Then the deflector is switched off until one ion has met the focal plane and has been transmitted to an ion detector which is put behind the cell instead of the microscope objective (Fig. 3(b)). The beam is stopped within 1.4 ls reaction time after ion detection. This reaction time is dominated by the traveling time of an ion from the deflector to the focal plane. The quality of the beam at the irradiation position in air has been measured by analysing a polycarbonate foil as a nuclear track detector which has been irradiated by a point matrix with a pitch
of 5 lm · 5 lm under similar conditions as the cells later on. After etching the foil in alcoholic base one sees each ion hit in the foil as a dark point under bright field illumination in the optical microscope (Fig. 4(a)). Assuming a regular grid, as marked in red in Fig. 4(a), the distribution of distances of the points from the grid can be plotted in x- and y-direction independently. These distributions are shown in Fig. 4(b) and (c). A full width half maximum of 0.55 lm in x-direction and 0.4 lm in ydirection is evaluated which is some factors better than reported earlier [9]. It is close to the resolution of conventional optical microscopy and is therefore in line with the requirements to study protein dynamics in cells by optical microscopy after targeting a certain place in the single cell.
4. Matrix irradiation and repair foci in cell nuclei Up to now, most of our biological experiments have been performed by irradiating regular patterns onto a field of 500 · 500 lm2 where cells are densely grown onto a 6 lm thick Mylar foil. Such a regular
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Fig. 5. Data of Immunofluorescence images of p53BP1 binding protein1 (p53BP1)s accumulation in a single HeLa cell nucleus irradiated by matrix irradiation (5 · 5 lm pitch). Raw images are acquired by three dimensional microscopy, the fluorescence images are processed using the deconvolution algorithm Huygens [12]. The images represent the 53BP1 accumulation under the view of four differenct angles. Although the regular structure of the matrix can be recognised, the whole structure is slightly deformed as well as the line structure of each single ion track.
pattern can be the 5 lm · 5 lm point matrix as it was used for the evaluation of the beam resolution. Due to the possibility of a fast scanning and recognition (up to 100 ions per second at the moment, 1000 ions per second possible in future) such patterns are written within a few minutes which is not possible with collimated ion beams. Several experiments deal with the observation of the accumulation of repair proteins in foci along the ion track by immuno-fluorescence techniques on those irradiated cells after a certain time of further cell growth. Cell growth conditions and cell treatment after irradiation is described in detail in [9]. Micrographs of the fluorescence signals from secondary antibodies marking c–H2AX (the phosphorilated histon H2AX) in a single cell nucleus are shown in Fig. 5. The figure shows four different angular views of one three dimensional data set from optical microscopy of a cell which has been fixed 1.5 h after irradiation. The individual line spots show the c–H2AX mainly arranged along
the 5 · 5 lm2 point matrix shot with single 100 MeV oxygen ions and show only minor deviation from the gross irradiation pattern. However, the lines are not really straight lines but show curvatures and a bulky structure which are not expected from angular deflections of the ions. The line in the middle is rather faded while the lines around show more or less a continuous structure. The different behaviour of line spots inside the cell nucleus and at the side as well as the line structure behaviour are commonly recognised and change with the time between irradiation and fixation. These data are subjected to an ongoing analysis in order to get a detailed knowledge on protein dynamics and cell repair.
5. Conclusion It has been demonstrated that the availability of a wide ion spectrum and high ion energies at a
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focused ion microprobe allow a new quality of experiments in radio-biology and in cell-biology in general. Fast scanning makes it possible to apply a certain irradiation pattern to the cells with many single shots. Heavy ions make enough damage that the track of a single ion is sufficient to see localised protein expression in the cell. It offers the opportunity to study repair dynamics in cells in three space coordinates beginning with well defined starting points in space and time.
Acknowledgement This work is supported by the Maier-LeibnitzLaboratorium der LMU und TU Mu¨nchen.
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