Radiation Protection Dosimetry Advance Access published April 24, 2015 Radiation Protection Dosimetry (2015), pp. 1–6
doi:10.1093/rpd/ncv192
ADVANCES IN MICROBEAM TECHNOLOGIES AND APPLICATIONS TO RADIATION BIOLOGY P. Barberet1,2,* and H. Seznec1,2 1 University of Bordeaux, CENBG, UMR 5797, Gradignan F-33170, France 2 CNRS, IN2P3, CENBG, UMR 5797, Gradignan F-33170, France *Corresponding author: [email protected]
INTRODUCTION Charged-particle microbeams (CPMs) are developed worldwide to perform targeted micro-irradiation of living cells. These facilities present unique features for radiation biology studies such as the possibility to target sub-cellular compartments with the micrometre precision, a precise dose control at the single-cell scale and an irradiation very well resolved in time. The rational for developing such devices was initially motivated by the necessity to study the cellular response to low doses of ionising radiation. Using broad beams or radioactive sources, the delivering of an average of one particle per cells leads to a Poisson distribution of the number of particle traversals. This means that 37 % of the cells receive no particle at all, 37 % receive one particle and 26 % receive more than one particle. Using a CPM, a single particle can be delivered uniformly to each cell, allowing a better understanding of the effects of environmental exposures where a cell is unlikely to receive more than one particle. Following the early work of Zirkle and Bloom in the 1950s(1), the first fully automated CPMs, developed in the Gray Laboratory (Northwood, UK)(2), at Pacific Northwest Laboratory (Washington, USA)(3) and at RARAF (Columbia University, USA)(4), were initially based on collimated beams delivered by Van de Graaff accelerators. These systems achieved a lateral resolution in air of a few micrometres and have been extensively used to study the cellular response to low doses of ionising radiation(5). More recently, CPMs formed by magnetic or electrostatic focusing have come into operation, allowing a better lateral resolution and a higher irradiation throughput by moving the beam from cell to cell using fast electromagnetic scanning
systems. By their ability to target the radiation into micrometre volumes inside a cell nucleus in a highly controlled way, CPMs are nowadays predominantly used to study DNA damage and repair(6). This is particularly true for high-LET CPMs, which provide the opportunity to investigate in cellulo DNA damage and repair following highly clustered damage. In order to perform these studies, CPMs are equipped with advanced fluorescence microscopy end-stations that allow the following of the early response to radiationinduced DNA damage (few seconds to minutes) at the single-cell scale online. Beside these applications, there is also a growing interest in the use of microbeams for ex vivo and in vivo irradiation, either in tissues or in small multicellular organisms. The different applications of CPMs have been reviewed previously(5, 6). A list of the currently active CPMs is provided in Table 1. The present review is following the last two editions of the international workshop on microbeam probes of cellular radiation response (New York 2012 and Bordeaux 2013) and is focusing on the latest applications to biological studies as well as the ongoing developments on microbeam end-stations. BIOLOGICAL EFFECTS OF SINGLE-ION TRACKS The first studies performed with CPMs began in the late 1990s at both Columbia University (New York, USA) and Gray Laboratory (Northwood, UK). The main breakthrough brought by these facilities was their ability to target single ions to individual cells. This unique feature was used to measure the effect of a single particle track on oncogenic transformation(17),
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Charged-particle microbeams (CPMs) allow the targeting of sub-cellular compartments with a counted number of energetic ions. While initially developed in the late 1990s to overcome the statistical fluctuation on the number of traversals per cell inevitably associated with broad beam irradiations, CPMs have generated a growing interest and are now used in a wide range of radiation biology studies. Besides the study of the low-dose cellular response that has prevailed in the applications of these facilities for many years, several new topics have appeared recently. By combining their ability to generate highly clustered damages in a micrometric volume with immunostaining or live-cell GFP labelling, a huge potential for monitoring radiation-induced DNA damage and repair has been introduced. This type of studies has pushed end-stations towards advanced fluorescence microscopy techniques, and several microbeam lines are currently equipped with the state-of-the-art time-lapse fluorescence imaging microscopes. In addition, CPMs are nowadays also used to irradiate multicellular models in a highly controlled way. This review presents the latest developments and applications of charged-particle microbeams to radiation biology.
P. BARBERET AND H. SEZNEC Table 1. Charged-particle microbeams for targeted irradiation of living cells. Laboratory
Particle p, a p p to Ca
IMP Fudan (China) CENBG Bordeaux (France) PTB Braunschweig (Germany) RIKEN Wako (Japan) SNAKE Munich (Germany)
p, a p, a p, a p, a p, a, Li to O, Si, Cl, I
GSI Darmstadt (Germany) JAERI Takasaki (Japan)
C to U rarely p, a, Li a, C, Ne, Ar
IMP Lanzhou (China)
C
Reference
1 –5 MeV 3.4 MeV p: 4 MeV a: 6 MeV O: 12 MeV 6 MeV 1 –3.5 MeV 2 –20 MeV 3 –4 MeV p: 4– 28 MeV a: 1.4– 10.5 MeV u21 Li– O: 1– 8 MeV u21 Si, Cl: 1– 4 MeV u21 I: 0.5– 2 MeV u21 1.4– 11.4 MeV u21 a: 12.5 MeV u21 C: 18.3 MeV u21 Ne: 13 and 17.5 MeV u21 Ar: 11.5 and 13.3 MeV u21 Several 100 MeV u21
(4) (7) (8)
(9) (10) (11) (12) (13)
(14) (15)
(16)
The list is constructed from the facilities reported at the last two microbeam workshops (period from 2010 to 2013).
cellular toxicity, nuclear DNA mutagenesis(18), micronuclei formation and genomic instability(19, 20). These studies showed that a single particle traversal has a considerable toxic and mutagenic effect and induces a significant increase of genomic instability. In addition, the authors found an oncogenic transformation efficiency significantly lower than the one associated with a Poisson distribution with an average of one alpha particle per nucleus. The authors suggested that multiple traversals dominate the biological response(17). Besides the studies performed with light ion microbeams, the investigation of the cellular response to single heavy ions has also been more recently started on several facilities(21). The heavy ion microbeam developed for targeted irradiation at GSI Darmstadt (14) has been used to measure the genomic instability and senescence/differentiation in descendants of fibroblasts exposed to exactly one or five carbon ions(22). This study showed that cells surviving single-ion traversal are often carrying clonal chromosome aberrations without chromosomal instability and undergo accelerated senescence. In this kind of studies, the main drawback of targeted CPMs irradiation is the limited number of cells that one can irradiate within an experiment. There has been a continuous effort to improve the irradiation throughput of microbeam, by speeding up the beam scanning systems or motorised stages. Another approach is the use, at microbeam end-stations, of sample manipulation techniques based on microfluidics. Promising results have been obtained at RARAF(23), and these methods can provide a way to increase drastically the irradiation
throughput by flowing the cells in micro-channels intersecting the beam path. BYSTANDER RESPONSES Since the beginning of their development, one of the main applications of CPMs to radiation biology has been the elucidation of non-targeted effects (such as bystander responses)(24). In particular, these facilities have been widely used to target a few cells in a population and study the response to their un-irradiated neighbours. Most of the studies have been conducted, using light ion microbeams, but the investigation of bystander responses following heavy ion irradiation has also started(21, 25, 26). A complete review on bystander responses studied with microbeams can be found in(27). RADIATION SENSITIVITY OF SUB-CELLULAR COMPARTMENTS The micrometre accuracy of CPMs and the exact control of the irradiation timing provide the opportunity to probe definite cellular response as a function of the sub-cellular target. Cytoplasmic irradiations The development of CPMs provides a unique way to study the radiation sensitivity of the sub-cellular compartments such as cytoplasm and mitochondrion. Since the first studies performed with CPMs, accumulating
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RARAF Columbia University (USA) SPICE NIRS Chiba (Japan) Ion Beam Center Surrey (UK)
Energy range
ADVANCES IN MICROBEAM TECHNOLOGIES
lines of evidence have shown that energy deposit by radiation in nuclear DNA is not an absolute requirement to trigger a damage and that extra-nuclear irradiation can induce biological effects. One of the first studies performed with CPMs concerned the radiation sensitivity of the cell cytoplasm(28). Indeed, Wu et al. showed that a targeted cytoplasmic irradiation with alpha particles leads to an increased level of mutations together with a lower cell killing when compared with nuclear irradiations(28). Since then, several studies have shown that cytoplasmic irradiations can induce 53BP1 radiation-induced foci in the nucleus as well as bystander responses [see review(29)].
The ability of CPMs to induce highly localised and clustered damage within a cell nucleus has recently opened new fields of investigation. By coupling microbeam irradiations with immunostaining, GFP-tagging, high-resolution fluorescence microscopy and time-lapse imaging, it became indeed possible to visualise the recruitment of proteins involved in DNA damage detection, signalling and repair. The first attempts to visualise microbeam-induced DNA damage took place 10 y ago in several laboratories: Gray Laboratory (Northwood, UK)(30), SNAKE (Munich, Germany)(31) and GSI (Darmstadt, Germany)(14). The visualisation of radiation-induced foci in targeted cells is currently extensively used both to validate the microbeam-targeting accuracy(7, 13, 14) and to study the cascade of signalling and repair proteins triggered by complex/clustered DNA damage(6). The main advantages of CPMs to perform such studies come from their high resolution in space, time and dose. Indeed, they allow to target precisely sub-nuclear structures, to draw geometrical irradiation patterns with the micrometre precision, to deliver ions with a precise timing and to control precisely the dose at the micrometre scale. Up to now, most of the data on kinetics of protein recruitment to damage sites have been obtained by laser micro-irradiation. In comparison, local irradiation with charged particles leads to complex DNA damage without the use of photo-sensitisers and a well-defined dosimetry. During the last 5 y, this application of microbeams has developed quickly. This is particularly the case using heavy ion microbeams. By using sequential pattern irradiation at SNAKE, Greubel et al. were able to show competition effects of the DNA repair proteins, Rad51 and 53BP1(32, 33). Taking advantage of the sub-micrometretargeting accuracy of the GSI heavy ion microbeam, Jakob et al. investigated the g-H2A.X foci formation within heterochromatin domains(34). Here, targeted micro-irradiation showed that H2A.X is early phosphorylated within heterchromatin, but the damage site is expelled from the centre to the periphery of chromocentres within 20 min. The GSI heavy ion microbeam has also been more recently used to target cell nuclei
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Nuclear irradiation: DNA damage and repair studies
with a counted number of ions to study H2A.X phosphorylation observed in undamaged chromatin over the whole-cell nucleus(35). Meyer et al. showed that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and to H2A.X phosphorylation. Besides the development of several microbeams at RARAF(36), several light ion microbeams have also evolved in the last years to allow DNA damage and repair studies with lower LETs (mainly protons). Konishi et al. have reported a new experimental facility (SPICE at National Institute of Radiological Science, Japan), allowing cell targeting with 3.4-MeV protons with an accuracy of 2 mm. This facility allows geometrical irradiations and is opened to external users(7). The Surrey vertical microbeam is becoming operational for targeted irradiation with ions ranging from protons to calcium(8). The authors recently updated their light ion microbeam at CENBG. This set-up is based on the first prototype developed for low-dose studies(37) on which the end-station has been fully rebuilt to improve its performance and to allow fast irradiation based on electrostatic beam scanning and time-lapse imaging online (submitted manuscript). Indeed, to go further in dynamic measurements of the cell response to ionising radiation, there is a great interest to couple microbeam irradiation and timelapse imaging. At present, five CPMs have been equipped with state-of-the-art fluorescence microscopes, allowing time-lapse imaging to be performed online. This experimental approach provides data on the kinetics of the DNA repair molecules at different LETs as well as on other responses. The possibility to perform live-cell imaging online has been initially demonstrated at GSI on a broad beam facility(38). Time-lapse acquisition has been then introduced to the GSI end-station initially to visualise intracellular calcium concentrations(39) and recently expanded to DNA repair studies(40). The possibility to follow online the protein recruitment by live-cell imaging has also been developed at SNAKE in Munich(13). This set-up has been recently applied successfully to measure the kinetics of DNA repair proteins at different LETs(41). Moreover, by combining a geometrical irradiation and live-cell imaging, Girst et al. have studied the chromatin mobility and shown a sub-diffusion behaviour of DNA double-strand breaks (DNA-DSBs)(42). A similar approach has been used at PTB Braunschweig where the live-cell imaging facility(11) has been used to compare the recruitment kinetics following proton and alpha-particle irradiation(43). A customised twophoton microscope has been also developed at the microbeam II end-station at RARAF to follow postirradiation cellular dynamics(44). These different facilities are used in a routine way to investigate the early dynamics of the cellular response to radiation damage. In parallel, new developments are still in progress. One can cite, for example, the pioneer work performed at
P. BARBERET AND H. SEZNEC
GSI to allow the use of photobleaching techniques in combination with high LET particle irradiation(40). Using this approach, protein turnover with little or no local concentration change can be made visible by selectively manipulating the fluorescence state of the tagged molecules in the volume of interest by means of laser pulses. This approach can provide precise measurements of the exchange and binding behaviour of repair proteins after targeted ion irradiation can then be obtained(45). TOWARDS THE USE OF MULTICELLULAR SPECIMENS
SUMMARY Charged-particle microbeam, initially developed for low-dose studies, is undergoing a rapid evolution to meet various biological applications. The improvement of their accuracy, throughput and the evolution of the end-stations towards advanced microscopy allows these microbeams to be used for DNA repair studies following complex radiation-induced damage. Investigation of the dynamics of the cellular early response to ionising radiation is also becoming possible thanks to the time-lapse imaging capabilities recently added to the microbeam end-stations. There is no doubt that these devices will continue to play an important a role in radiation biology. FUNDING This work has been supported by the European Community as an Integrating Activity “Support of Public and Industrial Research Using Ion Beam Technology (SPIRIT)” under the EC contract no 227012 and Marie Curie Actions “SPRITE”. The authors are supported by the CNRS (“Mission interdisciplinaire”, PRISM project). The AIFIRA facility is supported by the Region Aquitaine. REFERENCES 1. Zirkle, R. E. and Bloom, W. Irradiation of parts of individual cells. Science 117(80), 487 –493 (1953). 2. Folkard, M., Vojnovic, B., Prise, K. M., Bowey, A. G., Locke, R. J. et al. A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles. Int. J. Radiat. Biol. 72, 375–385 (1997). 3. Nelson, J. M., Brooks, A. L., Metting, N. F., Khan, M. A., Buschbom, R. L. et al. Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells. Radiat. Res. 145, 568–574 (1996). 4. Randers-Pehrson, G., Geard, C., Johnson, G., Elliston, C. and Brenner, D. The Columbia University single-ion microbeam. Radiat. Res. 156, 210–214 (2001).
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Data from in cellulo experiments on monolayers are very useful to understand fundamental mechanisms but are difficult to extrapolate to understand the in vivo response. Three-dimensional tissue models can be used in combination with microbeam irradiation to overcome these limitations. So far, most of the microbeam studies have been performed with reconstructed human epidermis(46 – 49). Organotypic slice culture methods have been also recently used as an ex vivo model for radiation biology applications as they mimic the tissues’ natural three-dimensional cytoarchitecture and they can be used to study the radiation effect on several kinds of human issues(50). Microbeam irradiations have also been extended to the irradiation of small multicellular models. Defined and well-characterised biological models are indeed compatible with the limited range of the particles used at CPMs. The first attempts to adapt chargedparticle microbeam irradiation to multicellular models have been performed at JAERI Takasaki. Sugimoto used the nematode Caenorhabditis elegans (C. elegans) to investigate tissue-specific, local biological response to radiation in organisms(51). On the same facility, Fukamoto et al. developed a specific experimental set-up to irradiate silkworms larvae (Bombyx mori) with heavy ions in the frame of radio-microsurgery studies(52). Microbeam irradiations of multicellular organism have also been carried out at other facilities. Choi et al. used the SPICE microbeam to investigate low-dose effects in zebrafish Danio rerio embryos. The main advantage of using zebrafish embryos as a model for radiation biology is that the human and zebrafish genomes share considerable homology, including conservation of most DNA repair-related genes(53, 54). RARAF at Columbia University used the C. elegans nematode to obtain important insights on non-targeted effects(55). Among the different multicellular models used at microbeams, the C. elegans nematode presents numerous advantages for in vivo investigation of radiation effects: simple culture conditions and maintenance, rapid life cycle, transparent body, adult organism has only 959 somatic cells and its anatomy is invariant from one animal to the next. Moreover, a wide variety of mutants and transgenic
strains are fully characterised and available at the C. elegans Genome Center (CGC). From a practical point of view, this model is small enough to be compatible with microbeam irradiation since the diameter of its body is 50 mm, and its length is 1 mm. Caenorhabditis elegans is also used as a biological model for space flight research, including the biological effects of cosmic radiation(56). The manipulation of living animals at microbeam end-station can nevertheless turn out to be puzzling as these models are often moving on their support. Here again, new developments in microfluidics are on the way to obtain high throughput and immobilisation of the animals during irradiation(57).
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38. Jakob, B., Rudolph, J. H., Gueven, N., Lavin, M. F. and Taucher-Scholz, G. Live cell imaging of heavy-ioninduced radiation responses by beamline microscopy. Radiat. Res. 163, 681–690 (2005). 39. Du, G., Fisher, B. E., Voss, K. O., Becker, G., TaucherSholz, G., Kraft, G. and Thiel, G. The absence of an early calcium response to heavy-ion radiation in mammalian cells. Radiat. Res. 170, 316–326 (2008). 40. Merk, B., Voss, K.-O., Mu¨ller, I., Fischer, B. E., Jakob, B. et al. Photobleaching setup for the biological end-station of the darmstadt heavy-ion microprobe. Nucl. Instrum. Methods Phys. Res. Sect. B 306, 81–84 (2013). 41. Hable, V., Drexler, G. A., Bru¨ning, T., Burgdorf, C., Greubel, C. et al. Recruitment kinetics of DNA repair proteins Mdc1 and Rad52 but Not 53BP1 depend on damage complexity. PLoS One 7, e41943 (2012). 42. Girst, S., Hable, V., Drexler, G. A., Greubel, C., Siebenwirth, C. et al. Subdiffusion supports joining of correct ends during repair of DNA double-strand breaks. Sci. Rep. 3, 2511 (2013). 43. Mosconi, M., Giesen, U., Langner, F., Mielke, C., Dalla Rosa, I. et al. 53BP1 and MDC1 foci formation in HT1080 cells for low- and high-LET microbeam irradiations. Radiat. Environ. Biophys. 50, 345–352 (2011). 44. Bigelow, A. W., Geard, C. R., Randers-Pehrson, G. and Brenner, D. J. Microbeam-integrated multiphoton imaging system. Rev. Sci. Instrum. 79, 123707 (2008). 45. Ishikawa-Ankerhold, H. C., Ankerhold, R. and Drummen, G. P. C. Advanced fluorescence microscopy techniques— FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17, 4047–4132 (2012). 46. Belyakov, O. V., Mitchell, S. A., Parikh, D., RandersPehrson, G., Marino, S. A. et al. Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away. Proc. Natl Acad. Sci. USA 102, 14203–14208 (2005). 47. Sedelnikova, O. A., Nakamura, A., Kovalchuk, O., Koturbash, I., Mitchell, S. A. et al. DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models. Cancer Res. 67, 4295–4302 (2007).
doi:10.1093/rpd/ncv192
ADVANCES IN MICROBEAM TECHNOLOGIES AND APPLICATIONS TO RADIATION BIOLOGY P. Barberet1,2,* and H. Seznec1,2 1 University of Bordeaux, CENBG, UMR 5797, Gradignan F-33170, France 2 CNRS, IN2P3, CENBG, UMR 5797, Gradignan F-33170, France *Corresponding author: [email protected]
INTRODUCTION Charged-particle microbeams (CPMs) are developed worldwide to perform targeted micro-irradiation of living cells. These facilities present unique features for radiation biology studies such as the possibility to target sub-cellular compartments with the micrometre precision, a precise dose control at the single-cell scale and an irradiation very well resolved in time. The rational for developing such devices was initially motivated by the necessity to study the cellular response to low doses of ionising radiation. Using broad beams or radioactive sources, the delivering of an average of one particle per cells leads to a Poisson distribution of the number of particle traversals. This means that 37 % of the cells receive no particle at all, 37 % receive one particle and 26 % receive more than one particle. Using a CPM, a single particle can be delivered uniformly to each cell, allowing a better understanding of the effects of environmental exposures where a cell is unlikely to receive more than one particle. Following the early work of Zirkle and Bloom in the 1950s(1), the first fully automated CPMs, developed in the Gray Laboratory (Northwood, UK)(2), at Pacific Northwest Laboratory (Washington, USA)(3) and at RARAF (Columbia University, USA)(4), were initially based on collimated beams delivered by Van de Graaff accelerators. These systems achieved a lateral resolution in air of a few micrometres and have been extensively used to study the cellular response to low doses of ionising radiation(5). More recently, CPMs formed by magnetic or electrostatic focusing have come into operation, allowing a better lateral resolution and a higher irradiation throughput by moving the beam from cell to cell using fast electromagnetic scanning
systems. By their ability to target the radiation into micrometre volumes inside a cell nucleus in a highly controlled way, CPMs are nowadays predominantly used to study DNA damage and repair(6). This is particularly true for high-LET CPMs, which provide the opportunity to investigate in cellulo DNA damage and repair following highly clustered damage. In order to perform these studies, CPMs are equipped with advanced fluorescence microscopy end-stations that allow the following of the early response to radiationinduced DNA damage (few seconds to minutes) at the single-cell scale online. Beside these applications, there is also a growing interest in the use of microbeams for ex vivo and in vivo irradiation, either in tissues or in small multicellular organisms. The different applications of CPMs have been reviewed previously(5, 6). A list of the currently active CPMs is provided in Table 1. The present review is following the last two editions of the international workshop on microbeam probes of cellular radiation response (New York 2012 and Bordeaux 2013) and is focusing on the latest applications to biological studies as well as the ongoing developments on microbeam end-stations. BIOLOGICAL EFFECTS OF SINGLE-ION TRACKS The first studies performed with CPMs began in the late 1990s at both Columbia University (New York, USA) and Gray Laboratory (Northwood, UK). The main breakthrough brought by these facilities was their ability to target single ions to individual cells. This unique feature was used to measure the effect of a single particle track on oncogenic transformation(17),
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Charged-particle microbeams (CPMs) allow the targeting of sub-cellular compartments with a counted number of energetic ions. While initially developed in the late 1990s to overcome the statistical fluctuation on the number of traversals per cell inevitably associated with broad beam irradiations, CPMs have generated a growing interest and are now used in a wide range of radiation biology studies. Besides the study of the low-dose cellular response that has prevailed in the applications of these facilities for many years, several new topics have appeared recently. By combining their ability to generate highly clustered damages in a micrometric volume with immunostaining or live-cell GFP labelling, a huge potential for monitoring radiation-induced DNA damage and repair has been introduced. This type of studies has pushed end-stations towards advanced fluorescence microscopy techniques, and several microbeam lines are currently equipped with the state-of-the-art time-lapse fluorescence imaging microscopes. In addition, CPMs are nowadays also used to irradiate multicellular models in a highly controlled way. This review presents the latest developments and applications of charged-particle microbeams to radiation biology.
P. BARBERET AND H. SEZNEC Table 1. Charged-particle microbeams for targeted irradiation of living cells. Laboratory
Particle p, a p p to Ca
IMP Fudan (China) CENBG Bordeaux (France) PTB Braunschweig (Germany) RIKEN Wako (Japan) SNAKE Munich (Germany)
p, a p, a p, a p, a p, a, Li to O, Si, Cl, I
GSI Darmstadt (Germany) JAERI Takasaki (Japan)
C to U rarely p, a, Li a, C, Ne, Ar
IMP Lanzhou (China)
C
Reference
1 –5 MeV 3.4 MeV p: 4 MeV a: 6 MeV O: 12 MeV 6 MeV 1 –3.5 MeV 2 –20 MeV 3 –4 MeV p: 4– 28 MeV a: 1.4– 10.5 MeV u21 Li– O: 1– 8 MeV u21 Si, Cl: 1– 4 MeV u21 I: 0.5– 2 MeV u21 1.4– 11.4 MeV u21 a: 12.5 MeV u21 C: 18.3 MeV u21 Ne: 13 and 17.5 MeV u21 Ar: 11.5 and 13.3 MeV u21 Several 100 MeV u21
(4) (7) (8)
(9) (10) (11) (12) (13)
(14) (15)
(16)
The list is constructed from the facilities reported at the last two microbeam workshops (period from 2010 to 2013).
cellular toxicity, nuclear DNA mutagenesis(18), micronuclei formation and genomic instability(19, 20). These studies showed that a single particle traversal has a considerable toxic and mutagenic effect and induces a significant increase of genomic instability. In addition, the authors found an oncogenic transformation efficiency significantly lower than the one associated with a Poisson distribution with an average of one alpha particle per nucleus. The authors suggested that multiple traversals dominate the biological response(17). Besides the studies performed with light ion microbeams, the investigation of the cellular response to single heavy ions has also been more recently started on several facilities(21). The heavy ion microbeam developed for targeted irradiation at GSI Darmstadt (14) has been used to measure the genomic instability and senescence/differentiation in descendants of fibroblasts exposed to exactly one or five carbon ions(22). This study showed that cells surviving single-ion traversal are often carrying clonal chromosome aberrations without chromosomal instability and undergo accelerated senescence. In this kind of studies, the main drawback of targeted CPMs irradiation is the limited number of cells that one can irradiate within an experiment. There has been a continuous effort to improve the irradiation throughput of microbeam, by speeding up the beam scanning systems or motorised stages. Another approach is the use, at microbeam end-stations, of sample manipulation techniques based on microfluidics. Promising results have been obtained at RARAF(23), and these methods can provide a way to increase drastically the irradiation
throughput by flowing the cells in micro-channels intersecting the beam path. BYSTANDER RESPONSES Since the beginning of their development, one of the main applications of CPMs to radiation biology has been the elucidation of non-targeted effects (such as bystander responses)(24). In particular, these facilities have been widely used to target a few cells in a population and study the response to their un-irradiated neighbours. Most of the studies have been conducted, using light ion microbeams, but the investigation of bystander responses following heavy ion irradiation has also started(21, 25, 26). A complete review on bystander responses studied with microbeams can be found in(27). RADIATION SENSITIVITY OF SUB-CELLULAR COMPARTMENTS The micrometre accuracy of CPMs and the exact control of the irradiation timing provide the opportunity to probe definite cellular response as a function of the sub-cellular target. Cytoplasmic irradiations The development of CPMs provides a unique way to study the radiation sensitivity of the sub-cellular compartments such as cytoplasm and mitochondrion. Since the first studies performed with CPMs, accumulating
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RARAF Columbia University (USA) SPICE NIRS Chiba (Japan) Ion Beam Center Surrey (UK)
Energy range
ADVANCES IN MICROBEAM TECHNOLOGIES
lines of evidence have shown that energy deposit by radiation in nuclear DNA is not an absolute requirement to trigger a damage and that extra-nuclear irradiation can induce biological effects. One of the first studies performed with CPMs concerned the radiation sensitivity of the cell cytoplasm(28). Indeed, Wu et al. showed that a targeted cytoplasmic irradiation with alpha particles leads to an increased level of mutations together with a lower cell killing when compared with nuclear irradiations(28). Since then, several studies have shown that cytoplasmic irradiations can induce 53BP1 radiation-induced foci in the nucleus as well as bystander responses [see review(29)].
The ability of CPMs to induce highly localised and clustered damage within a cell nucleus has recently opened new fields of investigation. By coupling microbeam irradiations with immunostaining, GFP-tagging, high-resolution fluorescence microscopy and time-lapse imaging, it became indeed possible to visualise the recruitment of proteins involved in DNA damage detection, signalling and repair. The first attempts to visualise microbeam-induced DNA damage took place 10 y ago in several laboratories: Gray Laboratory (Northwood, UK)(30), SNAKE (Munich, Germany)(31) and GSI (Darmstadt, Germany)(14). The visualisation of radiation-induced foci in targeted cells is currently extensively used both to validate the microbeam-targeting accuracy(7, 13, 14) and to study the cascade of signalling and repair proteins triggered by complex/clustered DNA damage(6). The main advantages of CPMs to perform such studies come from their high resolution in space, time and dose. Indeed, they allow to target precisely sub-nuclear structures, to draw geometrical irradiation patterns with the micrometre precision, to deliver ions with a precise timing and to control precisely the dose at the micrometre scale. Up to now, most of the data on kinetics of protein recruitment to damage sites have been obtained by laser micro-irradiation. In comparison, local irradiation with charged particles leads to complex DNA damage without the use of photo-sensitisers and a well-defined dosimetry. During the last 5 y, this application of microbeams has developed quickly. This is particularly the case using heavy ion microbeams. By using sequential pattern irradiation at SNAKE, Greubel et al. were able to show competition effects of the DNA repair proteins, Rad51 and 53BP1(32, 33). Taking advantage of the sub-micrometretargeting accuracy of the GSI heavy ion microbeam, Jakob et al. investigated the g-H2A.X foci formation within heterochromatin domains(34). Here, targeted micro-irradiation showed that H2A.X is early phosphorylated within heterchromatin, but the damage site is expelled from the centre to the periphery of chromocentres within 20 min. The GSI heavy ion microbeam has also been more recently used to target cell nuclei
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Nuclear irradiation: DNA damage and repair studies
with a counted number of ions to study H2A.X phosphorylation observed in undamaged chromatin over the whole-cell nucleus(35). Meyer et al. showed that a transient dose-dependent activation of the kinases occurring on complex DNA lesions leads to their nuclear-wide distribution and to H2A.X phosphorylation. Besides the development of several microbeams at RARAF(36), several light ion microbeams have also evolved in the last years to allow DNA damage and repair studies with lower LETs (mainly protons). Konishi et al. have reported a new experimental facility (SPICE at National Institute of Radiological Science, Japan), allowing cell targeting with 3.4-MeV protons with an accuracy of 2 mm. This facility allows geometrical irradiations and is opened to external users(7). The Surrey vertical microbeam is becoming operational for targeted irradiation with ions ranging from protons to calcium(8). The authors recently updated their light ion microbeam at CENBG. This set-up is based on the first prototype developed for low-dose studies(37) on which the end-station has been fully rebuilt to improve its performance and to allow fast irradiation based on electrostatic beam scanning and time-lapse imaging online (submitted manuscript). Indeed, to go further in dynamic measurements of the cell response to ionising radiation, there is a great interest to couple microbeam irradiation and timelapse imaging. At present, five CPMs have been equipped with state-of-the-art fluorescence microscopes, allowing time-lapse imaging to be performed online. This experimental approach provides data on the kinetics of the DNA repair molecules at different LETs as well as on other responses. The possibility to perform live-cell imaging online has been initially demonstrated at GSI on a broad beam facility(38). Time-lapse acquisition has been then introduced to the GSI end-station initially to visualise intracellular calcium concentrations(39) and recently expanded to DNA repair studies(40). The possibility to follow online the protein recruitment by live-cell imaging has also been developed at SNAKE in Munich(13). This set-up has been recently applied successfully to measure the kinetics of DNA repair proteins at different LETs(41). Moreover, by combining a geometrical irradiation and live-cell imaging, Girst et al. have studied the chromatin mobility and shown a sub-diffusion behaviour of DNA double-strand breaks (DNA-DSBs)(42). A similar approach has been used at PTB Braunschweig where the live-cell imaging facility(11) has been used to compare the recruitment kinetics following proton and alpha-particle irradiation(43). A customised twophoton microscope has been also developed at the microbeam II end-station at RARAF to follow postirradiation cellular dynamics(44). These different facilities are used in a routine way to investigate the early dynamics of the cellular response to radiation damage. In parallel, new developments are still in progress. One can cite, for example, the pioneer work performed at
P. BARBERET AND H. SEZNEC
GSI to allow the use of photobleaching techniques in combination with high LET particle irradiation(40). Using this approach, protein turnover with little or no local concentration change can be made visible by selectively manipulating the fluorescence state of the tagged molecules in the volume of interest by means of laser pulses. This approach can provide precise measurements of the exchange and binding behaviour of repair proteins after targeted ion irradiation can then be obtained(45). TOWARDS THE USE OF MULTICELLULAR SPECIMENS
SUMMARY Charged-particle microbeam, initially developed for low-dose studies, is undergoing a rapid evolution to meet various biological applications. The improvement of their accuracy, throughput and the evolution of the end-stations towards advanced microscopy allows these microbeams to be used for DNA repair studies following complex radiation-induced damage. Investigation of the dynamics of the cellular early response to ionising radiation is also becoming possible thanks to the time-lapse imaging capabilities recently added to the microbeam end-stations. There is no doubt that these devices will continue to play an important a role in radiation biology. FUNDING This work has been supported by the European Community as an Integrating Activity “Support of Public and Industrial Research Using Ion Beam Technology (SPIRIT)” under the EC contract no 227012 and Marie Curie Actions “SPRITE”. The authors are supported by the CNRS (“Mission interdisciplinaire”, PRISM project). The AIFIRA facility is supported by the Region Aquitaine. REFERENCES 1. Zirkle, R. E. and Bloom, W. Irradiation of parts of individual cells. Science 117(80), 487 –493 (1953). 2. Folkard, M., Vojnovic, B., Prise, K. M., Bowey, A. G., Locke, R. J. et al. A charged-particle microbeam: I. Development of an experimental system for targeting cells individually with counted particles. Int. J. Radiat. Biol. 72, 375–385 (1997). 3. Nelson, J. M., Brooks, A. L., Metting, N. F., Khan, M. A., Buschbom, R. L. et al. Clastogenic effects of defined numbers of 3.2 MeV alpha particles on individual CHO-K1 cells. Radiat. Res. 145, 568–574 (1996). 4. Randers-Pehrson, G., Geard, C., Johnson, G., Elliston, C. and Brenner, D. The Columbia University single-ion microbeam. Radiat. Res. 156, 210–214 (2001).
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Data from in cellulo experiments on monolayers are very useful to understand fundamental mechanisms but are difficult to extrapolate to understand the in vivo response. Three-dimensional tissue models can be used in combination with microbeam irradiation to overcome these limitations. So far, most of the microbeam studies have been performed with reconstructed human epidermis(46 – 49). Organotypic slice culture methods have been also recently used as an ex vivo model for radiation biology applications as they mimic the tissues’ natural three-dimensional cytoarchitecture and they can be used to study the radiation effect on several kinds of human issues(50). Microbeam irradiations have also been extended to the irradiation of small multicellular models. Defined and well-characterised biological models are indeed compatible with the limited range of the particles used at CPMs. The first attempts to adapt chargedparticle microbeam irradiation to multicellular models have been performed at JAERI Takasaki. Sugimoto used the nematode Caenorhabditis elegans (C. elegans) to investigate tissue-specific, local biological response to radiation in organisms(51). On the same facility, Fukamoto et al. developed a specific experimental set-up to irradiate silkworms larvae (Bombyx mori) with heavy ions in the frame of radio-microsurgery studies(52). Microbeam irradiations of multicellular organism have also been carried out at other facilities. Choi et al. used the SPICE microbeam to investigate low-dose effects in zebrafish Danio rerio embryos. The main advantage of using zebrafish embryos as a model for radiation biology is that the human and zebrafish genomes share considerable homology, including conservation of most DNA repair-related genes(53, 54). RARAF at Columbia University used the C. elegans nematode to obtain important insights on non-targeted effects(55). Among the different multicellular models used at microbeams, the C. elegans nematode presents numerous advantages for in vivo investigation of radiation effects: simple culture conditions and maintenance, rapid life cycle, transparent body, adult organism has only 959 somatic cells and its anatomy is invariant from one animal to the next. Moreover, a wide variety of mutants and transgenic
strains are fully characterised and available at the C. elegans Genome Center (CGC). From a practical point of view, this model is small enough to be compatible with microbeam irradiation since the diameter of its body is 50 mm, and its length is 1 mm. Caenorhabditis elegans is also used as a biological model for space flight research, including the biological effects of cosmic radiation(56). The manipulation of living animals at microbeam end-station can nevertheless turn out to be puzzling as these models are often moving on their support. Here again, new developments in microfluidics are on the way to obtain high throughput and immobilisation of the animals during irradiation(57).
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P. BARBERET AND H. SEZNEC 48. Zlobinskaya, O., Girst, S., Greubel, C., Hable, V., Siebenwirth, C. et al. Reduced side effects by proton microchannel radiotherapy: study in a human skin model. Radiat. Environ. Biophys. 52, 123–133 (2013). 49. Miller, J. H., Chrisler, W. B., Wang, X. and Sowa, M. B. Confocal microscopy for modeling electron microbeam irradiation of skin. Radiat. Environ. Biophys. 50, 365– 369 (2011). 50. Merz, F., Mu¨ller, M., Taucher-Scholz, G., Ro¨del, F., Sto¨cker, H. et al. Tissue slice cultures from humans or rodents: a new tool to evaluate biological effects of heavy ions. Radiat. Environ. Biophys. 49, 457–462 (2010). 51. Sugimoto, T., Dazai, K., Sakashita, T., Funayama, T., Wada, S. et al. Cell cycle arrest and apoptosis in Caenorhabditis elegans germline cells following heavyion microbeam irradiation. Int. J. Radiat. Biol. 82, 31– 38 (2006). 52. Fukamoto, K., Shirai, K., Sakata, T., Sakashita, T., Funayama, T. et al. Development of the irradiation method for the first instar silkworm larvae using locally targeted heavy-ion microbeam. J. Radiat. Res. 48, 247– 253 (2007). 53. Choi, V. W. Y., Konishi, T., Oikawa, M., Iso, H., Cheng, S. H. et al. Adaptive response in zebrafish embryos induced using microbeam protons as priming dose and Xray photons as challenging dose. J. Radiat. Res. 51, 657– 664 (2010). 54. Choi, V. W. Y., Yum, E. H. W., Konishi, T., Oikawa, M., Cheng, S. H. et al. Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons. J. Radiat. Res. 481, 475– 481 (2012). 55. Bertucci, A., Pocock, R., Randers-Pehrson, G. and Brenner, J. Microbeam irradiation of the C. elegans nematode. J. Radiat. Res. 50, A49 –A54 (2009). 56. Adenle, A., Johnsen, B. and Szewczyk, N. Review of the results from the International C. elegans first experiment (ICE-FIRST). Adv. Sp. Res. 44, 210–216 (2009). 57. Buonanno, M., Garty, G., Grad, M., Gendrel, M., Hobert, O. et al. Microbeam irradiation of C. elegans nematode in microfluidic channels. Radiat. Environ. Biophys. 52, 531–537 (2013).
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