Proton Microbeam Research Articles

In-air micro-PIXE chemical state analysis of phosphorus and sulfur using a new parallel-beam wavelength-dispersive X-ray emission spectrometer

The proton induced K X-ray emission spectra of several different phosphorus and sulfur compounds were recorded with the new parallel-beam wavelength dispersive X-ray emission spectrometer. The spectrometer was installed recently at the external proton microbeam at the J. Stefan Institute to perform high energy resolution in-air micro-PIXE analysis in the tender X-ray range. Reported measurements are used to probe the capabilities of the spectrometer to perform also chemical state analysis. For phosphorus measurements, where the energy resolution of the spectrometer is the highest, a clear correlation between measured energy position of the Kα emission line and the phosphorus formal oxidation state is obtained. Chemical contrast is increased further in the Kβ emission spectra reflecting the structure of occupied valence molecular orbitals defined by the first coordination shell around the central phosphorus atom. A reasonable chemical contrast is maintained also in the sulfur Kβ emission spectra, despite the fact that the energy resolution of the spectrometer declines with increasing X-ray energy. Finally, an example of phosphorus speciation in reference biological material is presented, demonstrating the feasibility of the new setup to extend the capabilities of in-air micro-PIXE analysis towards chemical speciation.

Production and measurement of MeV proton microbeams in atmospheric environment based on glass capillary

<sec>Traditionally, ion microbeam is produced by focusing or/and collimating to reduce the beam size to submicron level. The traditional setup for producing the microbeam consists of an expensive focusing and collimating system with a large space, based on electromagnetic fields. Meanwhile, the microbeam obtained through pure collimation of metal micro-tubes is limited by the fabrication processing, i.e. the size of beam spot is largely limited to a few microns and its manufacture is not as simple as that of a glass capillary. Inspired by early studies of the guiding effect, the use of inexpensive and easy-to-make glass capillaries as the tool for ion external microbeam production has become a new direction.</sec><sec>In this work, we use a glass capillary with an open outlet (108 μm in diameter), which serves as a vacuum differential and collimating component, to produce a 2.5 MeV-proton microbeam directly from the linear accelerator into the atmosphere for measurements. We measure the beam spot diameter and energy distribution of the microbeam as a function of the tilt angle of the capillary. We also conduct calculations and ion trajectory analysis on the scattering process of 2.5 MeV protons on the inner walls.</sec><sec>The measurement results show that when the tilt angle is around 0°, there are a direct transmission part that maintains the initial incident energy, and a scattering part with the energy loss in the microbeam. It is found that the proportion of directly transmitted protons and the beam spot size are highest near zero tilt angle. As the tilt angle increases, the beam spot diameter decreases; when the tilt angle is greater than the geometric angle, all the microbeams come from the scattering with the energy loss. The simulation combined with the ion trajectory analysis based on the scattering process can explain the experimental results. It is found that the large angle scattering determines the entire external microbeam spot, and the central region of the beam spot is composed of directly penetrating ions, whose size is determined by the geometric shape of the glass capillary, i.e. the outlet diameter and aspect ratio.</sec><sec>The natural advantage of producing external micobeames easily and inexpensively through glass capillaries is their relative safety and stable operation, and the last but not least point is to simply locate the microbeams on the sample without complex diagnostic tools. The microbeams are expected to be widely used in fields such as radiation biology, medicine, and materials.</sec>

Targeted nuclear irradiation with a proton microbeam induces oxidative DNA base damage and triggers the recruitment of DNA glycosylases OGG1 and NTH1

DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons.

Primary and Secondary Bystander Effects of Proton Microbeam Irradiation on Human Lung Cancer Cells under Hypoxic Conditions.

Tumor hypoxia is the most common feature of radioresistance to the radiotherapy (RT) of lung cancer and results in poor clinical outcomes. High-linear energy transfer (LET) radiation is a novel RT technique to overcome this problem. However, a limited number of studies have been elucidated on the underlying mechanism(s) of RIBE and RISBE in cancer cells exposed to high-LET radiation under hypoxia. Here, we developed a new method to investigate the RIBE and RISBE under hypoxia using the SPICE-QST proton microbeams and a layered tissue co-culture system. Normal lung fibroblast (WI-38) and lung cancer (A549) cells were exposed in the range of 06 Gy of proton microbeams, wherein only ~0.04-0.15% of the cells were traversed by protons. Subsequently, primary bystander A549 cells were co-cultured with secondary bystander A549 cells in the presence or absence of a GJIC and NO inhibitor using co-culture systems. Studies show that there are differences in RIBE in A549 and WI-38 primary bystander cells under normoxia and hypoxia. Interestingly, treatment with a GJIC inhibitor showed an increase in the toxicity of primary bystander WI-38 cells but a decrease in A549 cells under hypoxia. Our results also show the induction of RISBE in secondary bystander A549 cells under hypoxia, where GJIC and NO inhibitors reduced the stressful effects on secondary bystander A549 cells. Together, these preliminary results, for the first time, represented the involvement of intercellular communications through GJIC in propagation of RIBE and RISBE in hypoxic cancer cells.

Polymeric Membranes Doped with Halloysite Nanotubes Imaged using Proton Microbeam Microscopy.

Polymeric membranes are useful tools for water filtration processes, with their performance strongly dependent on the presence of hydrophilic dopants. In this study, polyaniline (PANI)-capped aluminosilicate (halloysite) nanotubes (HNTs) are dispersed into polyether sulfone (PES), with concentrations ranging from 0.5 to 1.5 wt%, to modify the properties of the PES membrane. Both undoped and HNT-doped PES membranes are investigated in terms of wettability (static and time-dependent contact angle), permeance, mechanical resistance, and morphology (using scanning electron microscopy (SEM)). The higher water permeance observed for the PES membranes incorporating PANI-capped HNTs is, finally, assessed and discussed vis-à-vis the real distribution of HNTs. Indeed, the imaging and characterization in terms of composition, spatial arrangement, and counting of HNTs embedded within the polymeric matrix are demonstrated using non-destructive Micro Particle Induced X-ray Emission (µ-PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques. This approach not only exhibits the unique ability to detect/highlight the distribution of HNTs incorporated throughout the whole thickness of polymer membranes and provide volumetric morphological information consistent with SEM imaging, but also overcomes the limits of the most common analytical techniques exploiting electron probes. These aspects are comprehensively discussed in terms of practical analysis advantages.

Guiding as a General Consequence of the Charged Particle Interaction with the Inner Surface of an Insulator Capillary—Guiding of 1 MeV Proton Microbeam through Polytetrafluoroethylene Macrocapillary

The transmission of energetic, 1 MeV proton microbeam through a single, cylindrical shaped, macrometer-sized polytetrafluoroethylene capillary was studied experimentally. The capillary axis was tilted with respect to the axis of the incident ion beam. The tilting, the aspect ratio of the capillary and the small beam divergence disabled the geometrical transmission of the beam through the target. The intensity, energy, deflection and charge state of the transmitted beam were investigated. We found that the pure guided transmission of a MeV/amu energy ion beam is observable. We clearly identified three completely different stages during the guiding process according to the measured energy distribution of transmitted particles. At the beginning the transmission intensity was low and only inelastic contributions with energy lower than 1 MeV were found in the spectrum. Later, in the second stage, the elastic peak appeared and became more and more significant. Finally, when the stable transmission evolved, only the elastic peak was present and the inelastic area was totally absent as a direct consequence of the ion guiding and as a result of the charged particle interaction with a charged inner surface of the insulator capillary.

Proton Microbeam Targeted Irradiation of the Gonad Primordium Region Induces Developmental Alterations Associated with Heat Shock Responses and Cuticle Defense in Caenorhabditis elegans.

We describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found.

The COX-2/PGE2 Response Pathway Upregulates Radioresistance in A549 Human Lung Cancer Cells through Radiation-Induced Bystander Signaling.

This study aimed to determine the mechanism underlying the modulation of radiosensitivity in cancer cells by the radiation-induced bystander effect (RIBE). We hypothesized that the RIBE mediates cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in elevating radioresistance in unirradiated cells. In this study, we used the SPICE-QST microbeam irradiation system to target 0.07-0.7% cells by 3.4-MeV proton microbeam in the cell culture sample, such that most cells in the dish became bystander cells. Twenty-four hours after irradiation, we observed COX-2 protein upregulation in microbeam-irradiated cells compared to that of controls. Additionally, 0.29% of the microbeam-irradiated cells exhibited increased cell survival and a reduced micronucleus rate against X-ray irradiation compared to that of non-microbeam irradiated cells. The radioresistance response was diminished in both cell groups with the hemichannel inhibitor and in COX-2-knockout cells under cell-to-cell contact and sparsely distributed conditions. The results indicate that the RIBE upregulates the cell radioresistance through COX-2/PGE2 intercellular responses, thereby contributing to issues, such as the risk of cancer recurrence.

Targeted Central Nervous System Irradiation with Proton Microbeam Induces Mitochondrial Changes in Caenorhabditis elegans.

Fifty percent of all patients with cancer worldwide require radiotherapy. In the case of brain tumors, despite the improvement in the precision of radiation delivery with proton therapy, studies have shown structural and functional changes in the brains of treated patients with protons. The molecular pathways involved in generating these effects are not completely understood. In this context, we analyzed the impact of proton exposure in the central nervous system area of Caenorhabditis elegans with a focus on mitochondrial function, which is potentially implicated in the occurrence of radiation-induced damage. To achieve this objective, the nematode C. elegans were micro-irradiated with 220 Gy of protons (4 MeV) in the nerve ring (head region) using the proton microbeam, MIRCOM. Our results show that protons induce mitochondrial dysfunction, characterized by an immediate dose-dependent loss of the mitochondrial membrane potential (ΔΨm) associated with oxidative stress 24 h after irradiation, which is itself characterized by the induction of the antioxidant proteins in the targeted region, observed using SOD-1::GFP and SOD-3::GFP strains. Moreover, we demonstrated a two-fold increase in the mtDNA copy number in the targeted region 24 h after irradiation. In addition, using the GFP::LGG-1 strain, an induction of autophagy in the irradiated region was observed 6 h following the irradiation, which is associated with the up-regulation of the gene expression of pink-1 (PTEN-induced kinase) and pdr-1 (C. elegans parkin homolog). Furthermore, our data showed that micro-irradiation of the nerve ring region did not impact the whole-body oxygen consumption 24 h following the irradiation. These results indicate a global mitochondrial dysfunction in the irradiated region following proton exposure. This provides a better understanding of the molecular pathways involved in radiation-induced side effects and may help in finding new therapies.

Design of 50 MeV proton microbeam based on cyclotron accelerator

Design of 50 MeV proton microbeam based on cyclotron accelerator

Stimulation of Nuclear Factor (Erythroid-Derived 2)-like 2 Signaling by Nucleus Targeted Irradiation with Proton Microbeam

Nuclear factor (erythroid-derived 2)-like 2 (NRF2), well-known as a master antioxidative response regulator in mammalian cells, is considered as a potential target for radiation protection and cancer therapy sensitization. We examined the response of NRF2 signaling in normal human lung fibroblast WI-38 cells to nucleus targeted irradiation by 3.4 MeV proton microbeam. Nucleus targeted irradiation stimulated the nucleus accumulation of NRF2 and the expression of its target gene, heme oxygenase 1 (HO-1). The nucleus accumulation of NRF2 increased from 3 h to 12 h post 500 proton irradiation. In the 500 protons range, higher number of protons resulted in increased NRF2 nucleus accumulation. Activating NRF2 with tert-butylhydroquinone reduced DNA double-strand break (DSB) formation in nucleus targeted irradiation by 15%. Moreover, ATM phosphorylation was found in nucleus targeted irradiation. Inhibiting ATM with ku55933 prevented NRF2 nucleus accumulation. Furthermore, nucleus targeted irradiation activated ERK 1/2, and ROS-ERK 1/2 signaling regulated NRF2 nucleus accumulation. Taken together, NRF2 signaling was activated by nucleus targeted irradiation and mitigated DNA DSB. The discovery of ATM and ERK 1/2 as upstream regulators of NRF2 signaling in nucleus targeted cells revealed new information regarding radiation protection.

Repair Kinetics of DSB-Foci Induced by Proton and α-Particle Microbeams of Different Energies.

In this work, the induction and repair of radiation-induced 53BP1 foci were studied in human umbilical vein endothelial cells irradiated at the PTB microbeam with protons and α-particles of different energies. The data were analyzed in terms of the mean number of 53BP1 foci induced by the different ion beams. The number of 53BP1 foci found at different times post-irradiation suggests that the disappearance of foci follows first order kinetics. The mean number of initially produced foci shows the expected increase with LET. The most interesting finding of this work is that the absolute number of persistent foci increases with LET but not their fraction. Furthermore, protons seem to produce more persistent foci as compared to α-particles of even higher LET. This may be seen as experimental evidence that protons may be more effective in producing severe DNA lesions, as was already shown in other work, and that LET may not be the best suited parameter to characterize radiation quality.

Tunable room temperature ferromagnetism in fullerene thin film induced by 1 MeV proton microbeam irradiation

We report tunable ferromagnetic properties of thin films of fullerene upon 1 MeV proton microbeam ion irradiation, by varying the ion fluence. Focused microbeam scanning of 1 MeV proton ions have been performed on the fullerene thin films. Consequently, a stable and significant ferromagnetic ordering at room temperature has been observed in the fullerene thin films. The proton microbeam irradiation induces a maximum magnetic ordering in fullerene with optimum dosage and subsequently higher fluence irradiations yield, diminishing effect upon the observed ferromagnetic ordering due to the higher degree of damages. The X-ray diffraction and Raman analysis confirm the damage due to proton microbeam irradiation. The reduction in the saturation magnetic moment induced, is very sharp as a result of exposure to the larger ion fluence. The approximate distance between defects has been simulated computationally, and its relation with observed ferromagnetism has been established. The irradiation has been performed at moderate ion flux (low microbeam current) to avoid the possible annealing effects of the ion irradiation.

Spectral-sensitive proton radiography of thin samples with the pixel detector Timepix3

We evaluate a high-resolution contrast-enhanced method for energy-sensitive radiography of thin samples with low-energy protons at the light-ion Tandetron accelerator of the NPI-CAS in Rez near Prague. We make use of the high-sensitivity of the hybrid semiconductor pixel detectors Timepix enabled by integrated per-pixel signal processing electronics. For this work we use the Timepix3 ASIC chip equipped with a 500 µm Si sensor operated with the fast data rate AdvaPix readout electronics interface. Measurements are performed in air with a 2.9 MeV proton microbeam on thin samples (<100 µm thick). As referential and testing sample we use a set of aluminum foils stacked into a closely packed assembly of varying layers of well-defined thickness. This and other samples were imaged and placed in front of the detector in transmission geometry. Radiographies were collected with focused beam (few mm size) and a microbeam (few µm size). The imaging principle is based on high-resolution spectrometry of single transmitted particles. Contrast is obtained by registration of small differences in the deposited energy of the proton after passing through the sample. This can be measured in wide-range by detailed spectral-tracking analysis of the pixelated clusters in the pixel detector. We examine and evaluate various cluster-track parameters sensitive for imaging contrast such as deposited energy, cluster area (number of pixels) and cluster height (maximum energy value of the pixels in the cluster). The position of interaction in the detector is registered in sub-pixel resolution down to few µm scale for the particles and geometry used. Radiographies are reconstructed based on these individual parameters imaged in image bins of adjustable size (few µm up to few tens of µm). The technique developed with different cluster parameters is presented together with evaluation of image contrast sensitivity on various types of samples and beam energies.

Polymeric Membranes Doped with Halloysite Nanotubes Imaged by Proton Microbeam Microscopy

Polymeric Membranes Doped with Halloysite Nanotubes Imaged by Proton Microbeam Microscopy

Hypoxia and Proton microbeam: Role of Gap Junction Intercellular Communication in Inducing Bystander Responses on Human Lung Cancer Cells and Normal Cells.

Radiation-induced bystander effect (RIBE) has been identified as an important contributing factor to tumor resistance and normal tissue damage. However, the RIBE in cancer and normal cells under hypoxia remain unclear. In this study, confluent A549 cancer and WI-38 normal cells were subjected to condition of hypoxia or normoxia, before exposure to high-LET protons microbeam. After 6 h incubation, cells were harvested and assayed for colony formation, micronucleus formation, chromosome aberration and western blotting. Our results show that there were differences of RIBE in bystander A549 and WI-38 cells under hypoxia and normoxia. The differences were also observed in the roles of HIF-1α expression in bystander A549 and WI-38 cells under both conditions. Furthermore, inhibition of gap junction intercellular communication (GJIC) showed a decrease in toxicity of hypoxia-treated bystander A549 cells, but increased in bystander WI-38 cells. These findings clearly support that GJIC protection of bystander normal cells from toxicity while enhancing in bystander cancer cells. Together, the data show a promising strategy for high-LET radiation in designing an entire new line of drugs, either increase or restore GJIC in bystander cancer cells which in turn leads to enhancement of radiation accuracy for treatment of hypoxic tumors.

Bias-dependent displacement damage effects in a silicon avalanche photodiode

Radiation effects studies on a commercial beveled-edge avalanche photodiode (APD) showed that applying reverse bias to the device during irradiation enhanced the severity of apparent damage. Proton microbeam irradiations were made using a proton beam energy of 2 MeV and fluence ranging from 2.0×1010 to 5.1×1012 cm−2. Charge collection measurements using the ion beam induced charge (IBIC) technique showed that relative losses increased by up to an order of magnitude when the reverse bias applied during irradiation increased from 50 to 1500 V. The presence of reverse bias also led to equivalent losses in charge collection that would only be seen in irradiations with an order of magnitude higher fluence in unbiased APDs. The results demonstrate that bias-enhanced irradiation damage is insufficiently understood and must be accounted for in characterizations of radiation effects.

The role of the construction and sensitive volume of compact ionization chambers on the magnetic field-dependent dose response.

The magnetic-field correction factors of compact air-filled ionization chambers have been investigated experimentally and using Monte Carlo simulations up to 1.5T. The role of the nonsensitive region within the air cavity and influence of the chamber construction on its dose response have been elucidated. The PTW Semiflex 3D 31021, PinPoint 3D 31022, and Sun Nuclear Cooperation SNC125c chambers were studied. The factors were measured at the experimental facility of the German National Metrology Institute (PTB) up to 1.4T using a 6 MV photon beam. The chambers were positioned with the chamber axis perpendicular to the beam axis (radial); and parallel to the beam axis (axial). In both cases, the magnetic field was directed perpendicular to both the beam axis and chamber axis. Additionally, the sensitive volumes of these chambers have been experimentally determined using a focused proton microbeam and finite element method. Beside the simulations of factors, detailed Monte Carlo technique has been applied to analyse the secondary electron fluence within the air cavity, that is, the number of secondary electrons and the average path length as a function of the magnetic field strength. A nonsensitive volume within the air cavity adjacent to the chamber stem for the PTW chambers has been identified from the microbeam measurements and FEM calculations. The dose response of the three investigated ionization chambers does not deviate by more than 4% from the field-free case within the range of magnetic fields studied in this work for both the radial and axial orientations. The simulated for the fully guarded PTW chambers deviate by up to 6% if their sensitive volumes are not correctly considered during the simulations. After the implementation of the sensitive volume derived from the microbeam measurements, an agreement of better than 1% between the experimental and Monte Carlo factors for all three chambers can be achieved. Detailed analysis reveals that the stem of the PTW chambers could give rise to a shielding effect reducing the number of secondary electrons entering the air cavity in the presence of magnetic field. However, the magnetic field dependence of their path length within the air cavity is shown to be weaker than for the SNC125c chamber, where the length of the air cavity is larger than its diameter. For this chamber it is shown that the number of electrons and their path lengths in the cavity depend stronger on the magnetic field. For clinical measurements up to 1.5T, the required corrections of the three chambers could be kept within 3% in both the investigated chamber orientations. The results reiterate the importance of considering the sensitive volume of fully guarded chambers, even for the investigated compact chambers, in the Monte Carlo simulations of chamber response in magnetic field. The resulting magnetic field-dependent dose response has been demonstrated to depend on the chamber construction, such as the ratio between length and the diameter of the air cavity as well as the design of the chamber stem.

Spatial and temporal distribution of a 1-MeV proton microbeam guided through a poly(tetrafluoroethylene) macrocapillary

We present computer simulations about the spatial and temporal evolution of a 1-MeV proton microbeam transmitted through an insulating macrocapillary with the length of 45 mm and with the inner diameter of 800 \ensuremath{\mu}m. The axis of the capillary was tilted to 1\ifmmode^\circ\else\textdegree\fi{} relative to the axis of the incident beam, which ensured geometrical nontransparency. The simulation is based on the combination of stochastic (Monte Carlo) and deterministic methods. It involves (1) random sampling of the initial conditions, according to distributions generated by the widely used and freely available computer software packages, srim and wintrax, (2) the numerical solution of the governing equations for following the classical trajectory of the projectiles, and (3) the description of the field-driven charge migration on the surface and in the bulk of the insulator material. We found that our simulation describes reasonably all of our previous experimental observations, indicating the functionality and reliability of the applied model. In addition, we found that at different phases of the beam transmission, different atomic processes result in the evolution of the beam distribution. First, in a scattering phase, the multiple small angle atomic scattering dominates in the beam transmission, resulting in an outgoing beam into a wide angular range and in a wide energy window. Later, in a mixed phase, scattering and guiding happens simultaneously, with a continuously increasing contribution of guiding. Finally, in the phase of the stabilized, guided transmission, a quadrupolelike focusing effect is observed, i.e., the transmitted beam is concentrated into a small spot, and the transmitted protons keep their initial kinetic energy.

DNA damage response in prostate cancer cells by proton microbeam irradiation.

BackgroundIn cancer radiotherapy, microbeam is an advanced and effective tool in investigating radiobiology. Currently, evidence to support the radiobiology of proton beam radiotherapy for prostate cancer is limited. This study aimed to investigate the DNA damage response of proton microbeam irradiation in prostate cancer.MethodsSingle-particle irradiation system to cells (SPICE) was used to perform the proton microbeam radiation-induced DNA damage response. The SPICE can deliver defined number of protons (3.4 MeV) to the cell nucleus. Different quantities of protons were irradiated to observe differential dose responses in prostate cancer cells. A total of 500 protons or defined proton doses were applied to PC-3 cell nucleus to investigate the kinetics of DNA double-strand breaks (DSB) repair after different time intervals; between 1 and 24 h post-irradiation. Subsequently, immunofluorescent staining of γ-H2AX was performed to detect DSB, and images were captured by immunofluorescence microscopy. Finally, γ-H2AX fluorescence intensity in each nucleus was quantified with Image J software.ResultsProton microbeam radiation-induced DSB were dependent on proton dose applied. After irradiated with 500 protons, relative expression levels of γ-H2AX were time dependent during DSB repair process. The γ-H2AX fluorescence intensity was maximum at 1 h post-irradiation. However, a gradual decrease was observed from 4 to 24 h.ConclusionsMicrobeam is a valuable tool for the exploration of DSB response. The findings of the present study show that microbeam irradiation targeted the nucleus with precision. This study is the first to reveal that immune-stained γ-H2AX assay with proton microbeam irradiation could predict DSB repair kinetics in PC-3 cells.