Adaptation of a neutron radiography instrument for live cell irradiation

Adaptation of a neutron radiography instrument for live cell irradiation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Adaptation of a neutron radiography instrument for live cell irradiation Nicholas Howell, Frederic Sierro, Raya Jarrah, Christopher Dobie, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5800297/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Neutron Capture Therapy (NCT) for cancer treatment is experiencing renewed interest due to advancements in accelerator-based neutron beams, treatment planning software, and patient positioning devices. This study presents the repurposing of an existing neutron radiography beamline (Dingo), at the OPAL research nuclear reactor, for radiobiological research and novel NCT development. Human glioblastoma cell cultures were irradiated for up to 10 mins with a flux of 2.57x10 8 n/cm 2 ⋅s (± 2.73x10 7 ) and the resulting impact was quantified by assessing DNA damage by both immunocytochemistry and flow cytometry. This low cost methodology extends the capability of an existing beamline to allow the development of novel neutron capture agents and study of neutron radiobiological mechanisms. Increasing availability of neutron sources for biological study in this fashion will accelerate the development of NCT for disease specific clinical application. Biological sciences/Cancer Biological sciences/Cancer/Cancer therapy Biological sciences/Cancer/Cancer therapy/Radiotherapy Biological sciences/Drug discovery/Drug screening Biological sciences/Cell biology/Cell death Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Neutron capture therapy (NCT) for the treatment of cancer is experiencing a resurgence of interest. This interest is being largely led by advances in compact accelerator-based neutron sources and appropriate beam shaping assemblies, decoupling this technique from expensive and unwieldy fission based sources, as well as advances in treatment planning software and patient positioning devices 1 . Broadly, NCT is an external beam radiotherapy modality that utilises neutron capture agents (NCA) - biochemically targeted stable isotopes of high neutron capture cross-section. Current methods approved for clinical use deliver 10 B isotope to cancerous tissue via metabolic accumulation 1 . Thermal neutron capture reaction in 10 B (Fig. 1 ) is the foundational principle of boron neutron capture therapy (BNCT) and results in the production of a high linear energy transfer (LET) α-particle (175 keV/µm), recoil 7 Li nucleus (150 keV/µm) and a prompt 478 keV γ-ray, via the following reaction 2 : These particles' short path length offer a theoretically very high targeting specificity, causing lethal damage to the tumour or other targeted cells while sparing surrounding healthy tissue. In Japan, NCT has been approved for treating recurrent head and neck cancers, and promising results have been observed in other cancers like melanoma and brain tumours 3 . This body of evidence, together with the technical advancements incorporating NCT with particle therapy, presents a strong case for further investigation, and a wider adoption, of NCT principles for the treatment of cancer. Additionally, recent work has demonstrated that biologically relevant and clinically useful neutron fluxes are generated internally during proton and heavy-ion particle therapy, as a by-product of the particle irradiation itself 4 , 5 . Referred to as Neutron Capture Enhanced Particle Therapy (NCEPT) 6 , this technology extends the principles of NCT to applications in particle therapy (proton and carbon ion) treatments. Exploiting this thermal neutron field that is internally generated within the patient could potentially lead to significant reductions in primary ion beam dose and associated fractions, that are expected to directly benefit patient outcomes and quality of life as well as provide treatment options for some poor prognosis cancers. In order to optimise and progress NCT research, access to thermal neutron radiation sources are essential. In general, the rate limiting step for most radiotherapy basic research and development is accessing appropriate radiation sources. Access to the limited beam time available for research on clinical instruments is highly competitive and the costs associated with running these machines greatly limits the total time available. A low cost, simple, neutron irradiation capability, that has minimal impact on standard operations, allows for the efficient, iterative experimentation required for novel pharmaceutical development and analysis of radiobiological mechanisms. This manuscript describes the repurposing of a pre-existing neutron imaging instrument (referred to as Dingo ) 7 , in use at the Open-Pool Australian Lightwater (OPAL) research nuclear reactor run by the Australian Nuclear Science and Technology Organisation, for the purposes of radiobiological research and novel NCT development. Dingo is currently the only thermal neutron beam available for research in Australia. The Dingo instrument is designed as a thermal neutron imaging instrument and its primary uses include palaeontology, materials science, archeology and industrial/engineering applications 7 . It is capable of neutron radiography and tomography and has two primary configurations: High resolution mode: 1 cm diameter pinhole producing a uniform field of 1.15x10 7 n/cm 2 ⋅s at the sample stage 8 . High intensity mode: 2 cm diameter pinhole producing 4.7x10 7 n/cm 2 ⋅s at the sample stage 8 . Even when operating in high-intensity mode the neutron flux at the sample stage is ∽10 lower than the minimum recommendation of 5x10 8 n/cm 2 ⋅s for reactor based BNCT 9 , 10 . The sample stage used for imaging sits 9.9 m from the primary shutter (Fig. 2). The inverse square law would indicate that halving the distance between the primary shutter and the sample could achieve a 4x increase in flux on the target, potentially increasing the flux to ∽2x10 8 n/cm 2 ⋅s, with further gains possible the closer to the primary shutter the sample can be located 11 . These increases, while still lower than the minimum recommended for BNCT, put the flux within a range that is usable for in vitro radiobiological and NCA structure-activity studies. An accessible location, immediately behind the tertiary shutter, was identified as a viable irradiation location. This positions the samples ∽4.8m from the primary shutter, effectively halving the distance, potentially generating the required increase in flux. As no sample stage is present at that location a mechanism to move the sample up to the tertiary shutter has to be developed. This system needs to efficiently and reproducibly position viable cell cultures at this location, which is at the entrance of the beam path to the instrument hutch on the opposite side of the 0.5 m thick concrete shielding wall. The dimensions of the tunnel through this wall, which sits in between the two helium flight tubes, is approximately 150 mm wide and 300 mm high. When repurposing existing infrastructure for radiobiology, successful live cell irradiation has three major requirements: The beam must be reliably located and its shape determined to allow effective targeting. The thermal neutron flux must be calculated so irradiation times can be determined and the fluence on the sample recorded. Cells must be placed into the path of the beam, while maintaining sterile conditions and viability, in a format suitable for the desired downstream analysis, for the correct period of time. In addition, this work builds off the successful development of a fully validated Geant4 Monte Carlo simulation model of the Dingo beamline 8 . This “digital twin” simulates the entire beampath and allows precise prediction of the neutron field. This model is essential for ensuring the efficient use of the beam for irradiation purposes. This model has been validated in-beam and out-of-beam using a combination of absolute and relative measurements, including neutron activation analysis, Bonner sphere spectroscopy and a real-time quad-MOSFET device 12 which are now able to be employed for quality assurance and validation purposes during radiobiological experimentation. The primary technique used for neutron flux measurements on the beamline is NAA of high-purity bare and cadmium covered gold foils. Gold is sensitive to thermal and also epithermal neutrons with high-magnitude peaks of 2.7x10 5 barn and 3.6x10 3 barn at 4.9 eV and 60.3 eV, respectively, and several few-hundred barn resonance peaks. Therefore, to measure the thermal neutron flux, gold is usually paired with a cadmium-covered foil or wire. Cadmium is a highly efficient thermal neutron absorber, and so its activity, or a reaction rate corresponding to the epithermal neutron capture, can be subtracted from the bare gold separating the thermal neutron flux 13 . This work demonstrates that simple, low cost repurposing of existing beamlines is a viable approach for establishing a basic neutron radiation biology capability and has resulted in the first thermal neutron source available for this kind of research in Australia. Materials and Methods Sample positioning The OPAL reactor supplies 15 instruments with neutron beams. However, one of its key applications is manufacturing of radiopharmaceuticals and neutron transmutation doping (NTD) of silicon. It requires inserting assemblies into the reactor core, which change the neutron scattering properties and may alter the spatial distribution of the neutron beams entering the beam port. Halving the distance between the pinhole and the samples increases not only the neutron flux, but also the variability in neutron intensities across the field, which is particularly important when operating in the high-intensity mode. Therefore, it is necessary to develop a robust and quick method to evaluate the relative position and planar distribution of the beam at Dingo. A simple solution for efficient estimation of the neutron field characteristics using EBT3 radiochromic films (Ashland) placed behind a 1.2 mm cadmium sheet was implemented. The cadmium sheet is wrapped in aluminium foil and the film affixed to the back via tape. This assembly is attached to the sample holder, placed into the relevant location and irradiated for approximately 10 min. Following irradiation, the cadmium sheet is removed leaving the film in place and left to self-develop for another 10 min to visualise the position of the beam relative to the sample holder. This enables the acquisition of an almost instant relative image of the thermal neutron field via the low energy γ-rays produced by neutron capture in cadmium, and ensures the samples are placed in the desired planar region of the field. Since the reaction rates in the polymer depend on the deposited energy, the “instant” optical signal drops with decreasing neutron flux incident on the cadmium sheet. Thus, to determine the semiquantitative profiles of the neutron beam intensity, the film can be removed and self-developed for ~ 12h to account for the non-uniform response. Finally, the film is scanned using a flatbed scanner to generate a 2D image. The optical density of the film is calculated from the pixel intensity which in turn is proportional to thermal neutron flux and can be plotted in ImageJ 14 . Thermal neutron flux Thermal neutron fluxes were quantified by gold neutron activation technique using the methodology covered in Jakubowski et al 17 . The gold wires were pre-weighed and positioned on 2 cm x 2 cm cassettes containing the biological samples that were arranged in a 3 x 3 grid. Samples were then aligned to the geometric centre of the beam using the aforementioned technique and irradiated for 8h. The characteristic γ-ray emission of 198 Au was measured with a high-purity Germanium detector (HPGe). In this work, we used bare gold wires adjacent to the samples and the average thermal to epithermal neutron ratios to determine the thermal neutron fluxes. These were previously measured with cadmium-paired gold foils distributed across the field in the same location at Dingo’s tertiary shutter 15 , 17 . Cell culture and Irradiation U87-MG (CellBank Australia, 89081402) and T98G (CellBank Australia, 92090213) cells were propagated in Minimum Essential Media (MEM, Gibco 11095098), supplemented with 10%FBS, 100U/ml penicillin and 100µg/ml streptomycin, at 37°C in a 5% CO 2 atmosphere. The day prior to irradiation 1x10 6 cells were seeded into a 24 well, optical base, tissue culture treated, assay plate to achieve ~ 80% confluence. Cultures were treated with 500uM [ 10 B]-BPA (> 98.4% 10 B, Interpharma Praha, Prague, Czech Republic), which was dissolved into cell media over a 2 hour time period at ~ 40°C in an ultrasonic water bath, and left to incubate for 2 hours. All the wells were filled with warm D-PBS to reduce headspace and then sealed with a sterile adhesive film. The sealed plates were placed in the vertical position on the sample holder mounted to a specially designed trolley (Fig. 3A). The sample was manoeuvred into the irradiation position (Fig. 3B) and irradiated for a period of 1 min or 10 min, achieving an estimated fluence of 1.5x10 10 n/cm 2 and 1.5x10 11 n/cm 2 respectively. Immunofluorescence Following irradiation, cells were incubated for 40 mins at 37°C, 5% CO 2 after which they were fixed in 10% NBF and stored at 4°C in PBS until processing. The U87-MG and T98G cells were washed with PBS, and permeabilized with 0.1% Triton-X for 10 minutes at room temperature on a rocker. Samples were blocked with 5% BSA for 1h at room temperature on a rocker. Cells were then incubated with primary antibody (γH2AX anti-mouse (Merk Life Science, Australia) 1:1200 dilution in 1% BSA) overnight at 4°C, washed with 0.1% PBST, and incubated with secondary antibody (Alexa Fluor 488 goat anti-mouse (AbCam, Australia) 1:800 dilution in 1% BSA) for 1h at room temperature on a rocker. After a final PBST wash, cells were first counterstained with Phalloidin iFluor-594(1:1000, Abcam, Australia) for 1h and DAPI (4′,6-diamidino-2-phenylindole) (1 µg/ml) for 20 min at room temperature, washed again, and mounted using FluoroShield mounting media (Sigma). Samples were left to cure overnight at room temperature (protected from light) and stored at 4°C for up to two weeks prior to analysis. The resulting γH2AX foci were quantified by counting the number of foci per nucleus in 55 randomly selected cells, by eye, using a 100x objective. Images of foci were also taken using a 63x objective. Focal stacks of representative regions were acquired using a Zeiss Imager Z2 microscope and deconvolved using the Zen deconvolution package. Maximum intensity projections were prepared from the deconvolved stacks to illustrate the distribution of foci within the nucleus. Flow cytometry At 60 min post irradiation, cells were washed with PBS and incubated in non-enzymatic cell dissociation solution (Sigma #C5914) for 5 to 10 min and kept on ice. Recovered cells were washed and stained with DAPI (0.1µg/ml) followed by an additional 2 PBS washes. Cells were then permeabilized and stained with γH2AX (Phospho-Histone H2A.X (Ser139) (CR55T33), PE, eBioscience™) antibody using the BD (Becton-Dickinson) Pharmingen™ Transcription Factor Buffer Set (#562574) following manufacturers’ recommendations. Acquisition was performed on a 5 lasers BD FACSymphony™ A3 Cell Analyzer. A minimum of 10000 DAPI neg cells were acquired per sample. Analysis was carried out using FlowJo ® software. Statistical analysis The Mann-Whitney U test was employed to analyse differences between groups in visualised γH2AX foci data. Visual analysis of the data showed foci per nucleus demonstrated a potentially skewed nature within this cell population. This skewing is due to the high background of spontaneous foci formation often present in cancer cells. This test was used as it compares medians and is sensitive to distribution differences, therefore able to provide insights into how these treatments differ. Results As previously demonstrated by Jakubowski et al., the neutron beam at Dingo constitutes approximately 59% thermal, 21% epithermal and 20% fast neutrons 17 . Imaging of the beam with radiochromic films shows an approximate square shape (Fig. 3). The x axis shows a relatively flat profile with sharp shoulders and fluxes slightly skewed to the left. The y axis profile has rounded shoulders and a more pronounced drop off in thermal neutron flux towards the top of the beam. The relatively flat area available for sample irradiation at the irradiation position is 10 cm in the x axis and 6 cm in the y axis with the distance in y being limited by the rounded shoulders. Location in the tunnel is determined relative to the sample holder, allowing the samples to be reliably and reproducibly placed into this location. The relative thermal neutron spatial distribution shows good agreement with the results of the absolute thermal neutron flux measurements using gold wires (Fig. 4). The 8 x 8 cm array, positioned in the centre of the beam, measured a neutron flux of 2.57x10 8 n/cm 2 ⋅s (± 2.73x10 7 ). The number of γH2AX foci per nucleus increased significantly with the addition of 500µM [ 10 B]-BPA when compared to irradiation alone demonstrating the presence of a neutron capture dose (Fig. 5). A Mann-Whitney U test was implemented to compare foci/nucleus between the 10min irradiation groups with and without the addition of [ 10 B]-BPA. There was a significant difference (U = 867.5, p < 0.0001) between the untreated (median = 8.5 (LL = 8.112, UL = 10.23), n = 55) and treated (median = 12.0 (LL = 11.38, UL = 14.26), n = 55) cells. A lower significance change (U = 969.5, p = 0.0010) was observed when comparing the 1 minute irradiation group (median = 12.0 (LL = 10.85, UL = 14.02), n = 55). Quantification of foci in this manner was complicated in these cell lines by the presence of a very high background of foci production, which can often be seen in cancer cell lines 16 . The background foci per nucleus of these cultures is around 10 and results in non-normally distributed data. This technique of counting foci also has limitations at higher doses with the foci becoming too close together to reliably distinguish one from the other leading to inaccuracy in counting. Deconvolved z-stack images of these cultures demonstrate the nature of γH2AX staining observed during the counting procedure (Fig. 6). A visual increase in both foci number and fluorescence intensity can be observed that correlates with the time of irradiation. Cells treated with 500µM [ 10 B]-BPA show increased levels of foci formation before tending to high levels of pan-nuclear staining following the 10 min irradiation. This increase in fluorescence intensity along with foci formation was further investigated by flow cytometry (Fig. 7). It showed an irradiation time dependent increase in fluorescence intensity in response to staining for γH2AX, following incubation with 500 µM [ 10 B]-BPA. A minor subset of cells exhibiting γH2AX staining is present in both [ 10 B]-BPA and NT controls that increases with irradiation time, becoming the majority of the population in the cultures treated with [ 10 B]-BPA following 10 mins of neutrons. Discussion To the best of our knowledge, this is the first, and only, neutron beam available for radiation biology research in Australia. This work demonstrates that low cost, repurposing of existing beamlines is a viable approach for the establishment of a neutron radiation biology capability. No permanent alterations to the existing infrastructure were required to achieve the successful irradiation of viable human tissue cultures at biologically significant dose rates. By utilising commercially available radiochromic films, we employed a robust and almost instant method to evaluate potential changes to the relative position and spatial distribution characteristics of the thermal neutron component of the beam at Dingo, which can occur due to modifications of the reactor core configuration. This approach increases experimental reproducibility and improves the positional accuracy of the samples. The thermal neutron flux can be effectively measured using gold NAA technique. Measurements of the thermal neutron flux at the irradiation position are in good agreement with the previously published fully validated Monte Carlo model of the beam 8 . Reported thermal neutron flux when running in high intensity mode at the sample stage is 4.7x10 7 n/cm 2 ⋅s. It accounts for approximately 59% of the neutron beam while the epithermal and fast neutron components comprise 21% and 20%, respectively. By approximately halving the distance to the primary shutter, the thermal neutron flux increases by nearly the factor of 4. It can be further increased by placing the samples immediately behind the tertiary shutter entry reaching 2.52x10 8 (± 2.73x10 7 ) n/cm 2 ⋅s. A previous experimental irradiation of tissue cultures at the Dingo sample stage used the 4.7x10 7 n/cm 2 ⋅s flux to deliver 1.2x10 12 n/cm 2 over a 7h time period 17 . Cells were kept in zero headspace at room temperature for the duration, which can introduce major confounding factors to the analysis of cell survival and proliferation. Repeating this experiment with methodology described above would result in a reduction of irradiation times to ~ 1.3h, with further reductions possible if the irradiation is restricted to the highest flux portion of the beam. Viable human tissue cultures placed behind the tertiary shutter and irradiated for periods of up to 10 min; the cultures were successfully recovered and the resulting activation of HR and NHEJ DNA repair pathways measured by γH2AX immunocytochemistry and flow cytometry. Significant increases in DNA damage were observed from the inclusion of 500µM 10 B-BPA, demonstrating the successful application of thermal neutron radiation to these samples. There are two major limitations that must be taken into consideration when irradiating tissue cultures as described above: The horizontal geometry of the beam requires that tissue cultures be held in a vertical position and; Irradiations are conducted under ambient conditions. To address point 1 all irradiations occur within air tight sealed containers, such as a 24 well plate with an adhesive seal or a screw top flask. To prevent cultures dehydrating during setup and irradiation time all containers are prepared with as close to zero headspace as practically achievable. In order to reduce the impacts of both points it is important to minimise the time the cultures are exposed to these conditions. Efficient sample setup and retrieval is key to keeping the impacts of these factors consistent between irradiations. The significance of these factors needs to be taken into consideration on a per cell line basis for future work. Inclusion of temperature control to the irradiation setup would be a valuable addition to this technique as it would allow for maintaining conditions in the cell cultures that are closer to physiological. Compact heating devices, such as Peltier heat exchanges that can be thermostatically controlled are being investigated. The complexities of placing such devices into such a neutron rich environment need to be taken into consideration from a functional and longevity perspective as well as the potential activation of the components. The current irradiation setup is constructed entirely out of plastic which does not activate to any significant extent under these conditions. The gold neutron activation analysis technique used in this study is the ‘gold standard’ for calculating thermal neutron flux coming from nuclear reactors. As this technique directly measures the activation of 198 Au via gamma ray spectrometry, the quantification of the thermal neutron flux is straightforward and robust. The technique itself is time-consuming compared to the short irradiation times of 1 min or less used for the irradiation of biological samples, with results being received a minimum of 24 h post experiment. It also requires sufficient gold activation for reliable quantification of the gamma ray emission and calculation of thermal neutron fluxes. The addition of a real-time neutron monitoring device that can distinguish between thermal and epithermal neutrons would be a very valuable addition. A device using a combination of MOSFET detectors covered with either boron carbide, cadmium and boron carbide, or polyethylene converters has been tested in simulation with very promising results 12 . Since the neutron beam properties can vary between the measurements, real-time feedback will be extremely valuable to allow optimisation of experimental protocols to more efficiently utilise the available beamtime and improve both the precision, and accuracy of irradiations. Conclusion The primary rate limiting factor for NCT research and development is access to appropriate radiation sources. Typically, hours on such instruments are limited and the merit based access models extremely competitive. To accelerate development of novel NCAs and targets, more beamtime is required. We have developed the techniques necessary to repurpose Dingo, a thermal neutron imaging instrument the OPAL reactor in Australia, to conduct biological irradiations for the purposes of radiobiological research and development. Sample positioning and techniques for robust beam characteristics evaluation and neutron flux measurements were developed to provide a radiobiological irradiation capability with no major changes to existing infrastructure. Opportunities for further development to improve precision and accuracy have been identified. It is hoped that by encouraging others to explore the potential of existing nuclear reactor-based facilities for biological research we can accelerate development in the field in order to meet the demand, and take full advantage, of the lower cost, modern accelerator-based neutron sources and techniques to utilise internally generated neutrons - a by-product of particle therapy treatment that will be available in the near future. Declarations Author Contribution N.H, F.S and R.J. conducted all biological experiments.C.D synthesised and provided all chemical compounds and assisted with methodological development.J.B ad U.G provided technical support, and access for all beamline operations.K.J and M.S provided the Monte Carlo model and support for physics measurements.N.H, F.S and K.J prepared the manuscript.The manuscript was reviewed by all authors. Acknowledgement Atila Stopic - gamma ray spectroscopy analysis of neutron activated gold.Deborah Wakeham - assistance with process development and logistics.Luca Daveos - assistance with design and engineering of sample holder.Australian Centre for Neutron Scattering (ACNS) - for availability of Dingo via access grants P16726, 17623 and 18678, and for supplying regular beamtime for work that falls well outside the scope of regular operations. Data Availability All data generated and analysed in this publication is available from the corresponding author upon request. References Boron neutron capture therapy. Current status and future perspectives - Dymova–2020 - Cancer Communications - Wiley Online Library. https://onlinelibrary.wiley.com/doi/10.1002/cac2.12089 Barth, R. F., Coderre, J. A., Vicente, M. G. H. & Blue, T. E. Boron Neutron Capture Therapy of Cancer: Current Status and Future Prospects. Clin. Cancer Res. 11 , 3987–4002 (2005). Matsumura, A. et al. Initiatives Toward Clinical Boron Neutron Capture Therapy in Japan. Cancer Biother Radiopharm . 38 , 201–207 (2023). Howell, N. et al. Neutron Capture Enhances Dose and Reduces Cancer Cell Viability in and out of Beam During Helium and Carbon Ion Therapy. Int. J. Radiat. Oncol. 10.1016/j.ijrobp.2024.02.052 (2024). Shiba, S. et al. Increased cell killing effect in neutron capture enhanced proton beam therapy. Sci. Rep. 14 , 28484 (2024). Safavi-Naeini, M. et al. Opportunistic dose amplification for proton and carbon ion therapy via capture of internally generated thermal neutrons. Sci. Rep. 8 , 16257 (2018). Salvemini, F., Bevitt, J., Liss, K. D. & Garbe, U. DINGO – the neutron imaging station at ANSTO: embracing material science, palaeontology, and cultural heritage. Neutron News . 27 , 14–19 (2016). Jakubowski, K. et al. A Monte Carlo model of the Dingo thermal neutron imaging beamline. Sci. Rep. 13 , 17415 (2023). Kumada, H., Sakae, T. & Sakurai, H. Current development status of accelerator-based neutron source for boron neutron capture therapy. EPJ Tech. Instrum. 10 , 1–15 (2023). IAEA. Current Status of Neutron Capture Therapy (INTERNATIONAL ATOMIC ENERGY AGENCY, 2001). Moghadam, S. R., Davani, F. A. & Danaei, M. Effective center measurement of long counter and calibration 241Am Be neutron source. Radiat. Prot. Environ. 36 , 90 (2013). Jakubowski, K. et al. Computational design and evaluation of a quad-MOSFET device for quality control of therapeutic accelerator-based neutron beams. Radiat. Meas. 177 , 107253 (2024). Dosimetry for Criticality Accidents . (INTERNATIONAL ATOMIC ENERGY AGENCY, Vienna, (1982). Fiji. an open-source platform for biological-image analysis | Nature Methods. https://www.nature.com/articles/nmeth.2019 Szieberth, M. & Zslonay, E. Determination of the Thermal Neutron Flux in the Core of the Reacto. (2012). Mirzayans, R. et al. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression. Int. J. Mol. Sci. 16 , 11609–11628 (2015). Kang, W. et al. Cyclic-RGDyC functionalized liposomes for dual-targeting of tumor vasculature and cancer cells in glioblastoma: An in vitro boron neutron capture therapy study. Oncotarget 8 , 36614–36627 (2017). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Feb, 2025 Reviews received at journal 15 Feb, 2025 Reviews received at journal 30 Jan, 2025 Reviewers agreed at journal 25 Jan, 2025 Reviewers agreed at journal 21 Jan, 2025 Reviewers invited by journal 20 Jan, 2025 Editor assigned by journal 20 Jan, 2025 Editor invited by journal 18 Jan, 2025 Submission checks completed at journal 16 Jan, 2025 First submitted to journal 09 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5800297","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":409333952,"identity":"0458246a-7387-4b66-baad-21703dbe3095","order_by":0,"name":"Nicholas Howell","email":"data:image/png;base64,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","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":true,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Howell","suffix":""},{"id":409333953,"identity":"cee3ddb8-2f9e-4246-8904-5ddeb501f5fe","order_by":1,"name":"Frederic Sierro","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Frederic","middleName":"","lastName":"Sierro","suffix":""},{"id":409333954,"identity":"ae814cba-12ca-4683-b4f2-0f8726c5a6a2","order_by":2,"name":"Raya Jarrah","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Raya","middleName":"","lastName":"Jarrah","suffix":""},{"id":409333955,"identity":"872ab680-ae61-4b62-9d37-e9a56c6cf7d7","order_by":3,"name":"Christopher Dobie","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Dobie","suffix":""},{"id":409333956,"identity":"7a602663-074a-4ca7-828d-a69c4a6925d8","order_by":4,"name":"Joseph J. Bevitt","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"J.","lastName":"Bevitt","suffix":""},{"id":409333957,"identity":"f3637e64-9975-4545-a315-7cf96507a912","order_by":5,"name":"Ulf Garbe","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Ulf","middleName":"","lastName":"Garbe","suffix":""},{"id":409333958,"identity":"417c23b4-c6a1-4537-bec7-645de6b6a417","order_by":6,"name":"Klaudiusz Jakubowski","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Klaudiusz","middleName":"","lastName":"Jakubowski","suffix":""},{"id":409333960,"identity":"a8686bb0-2d5f-48aa-994f-182862adf756","order_by":7,"name":"Mitra Safavi-Naeini","email":"","orcid":"","institution":"Australian Nuclear Science and Technology Organisation","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Safavi-Naeini","suffix":""}],"badges":[],"createdAt":"2025-01-10 03:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5800297/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5800297/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-02382-4","type":"published","date":"2025-05-22T15:58:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75409215,"identity":"66dd727e-6982-4e01-8391-cbb516da0662","added_by":"auto","created_at":"2025-02-04 09:03:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7468,"visible":true,"origin":"","legend":"\u003cp\u003eSchematics of the neutron capture reaction in \u003csup\u003e10\u003c/sup\u003eB.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/418fbbc231348ca7480f5531.jpg"},{"id":75407691,"identity":"7949d04a-3efb-4dd8-8519-19b9c00048a4","added_by":"auto","created_at":"2025-02-04 08:55:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":68635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram of the Dingo beamline \u003ca href=\"https://www.zotero.org/google-docs/?GAmkoW\"\u003e\u003csup\u003e8\u003c/sup\u003e\u003c/a\u003e. SS = secondary shutter; TS = tertiary shutter ; FT = flight tube; PFBS = pre-flight tube beam slits; FRM = floor rail mounting; DB = detector box; BS = beam stop. The relative positions of the sample stage and irradiation position are indicated. (\u003cstrong\u003eB) \u003c/strong\u003eSample mount designed for holding tissue culture plates vertically positioned within the neutron beam at the irradiation position. Samples are loaded onto this mount before being driven up to the irradiation position immediately behind the tertiary shutter. The modular design allows for various sample formats to be mounted and positioned for irradiation.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/a2425ab49895438f3c804ba8.jpg"},{"id":75405414,"identity":"2a0030d0-56ba-4c16-a6f5-1df2d770f118","added_by":"auto","created_at":"2025-02-04 08:47:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106640,"visible":true,"origin":"","legend":"\u003cp\u003eThermal neutron field characteristics measured with the radiochromic film (\u003cstrong\u003eA\u003c/strong\u003e). The use of this method allows for a quick and accurate sample alignment with the beam. Line profiles, when taken through the centre, demonstrate the slightly skewed nature of thermal neutron flux within the beam. In the X profile (\u003cstrong\u003eB\u003c/strong\u003e) the beam intensity can be seen to drop off slightly from left to right but remains linear until it falls away sharply at the edge. The Y profile (\u003cstrong\u003eC\u003c/strong\u003e) shows a flatter profile but with much more pronounced shoulders.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/385163c9e530e81b6048eaa0.jpg"},{"id":75405417,"identity":"5bc4990b-db4e-4cd7-968d-8cc7a2ede52d","added_by":"auto","created_at":"2025-02-04 08:47:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":28122,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial distribution of the thermal neutron field measured using gold wire activation technique. The variation in neutron flux across the field has been estimated to be approximately 2.73x10\u003csup\u003e7\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e⋅s.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/1dc7f37e468e41d87f631283.jpg"},{"id":75407698,"identity":"622f3129-ba90-46da-852e-d101886a80ec","added_by":"auto","created_at":"2025-02-04 08:55:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20003,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of γH2AX foci per nucleus in U87MG cells following 1 min and 10 min of neutron irradiation. Statistical analysis using the Mann-Whitney U test showed a significant difference between non-treated (NT) and 500uM [\u003csup\u003e10\u003c/sup\u003eB]-BPA treated cells (1min; p=0.0010, 10min; p\u0026lt;0.0001). No significance is observed from the 1 min irradiation time in this cell line. This shows a measurable impact of neutron capture as a result of irradiation that is significantly increased from just neutron irradiation alone.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/df79bac8f21c6a193496bb80.jpg"},{"id":75405440,"identity":"3b84c5bf-2016-4203-85fe-4fa20b474970","added_by":"auto","created_at":"2025-02-04 08:47:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":170825,"visible":true,"origin":"","legend":"\u003cp\u003eImmunocytochemistry analysis of γH2AX foci formation in T98G cells in response to neutron irradiation in the presence or absence of 500 μM [\u003csup\u003e10\u003c/sup\u003eB]-BPA. When [\u003csup\u003e10\u003c/sup\u003eB]-BPA was present, an increase in γH2AX foci formation was observed following 1 minute of neutron irradiation. At 10 min, many cells began to exhibit a pan-nuclear response, which was not present in the cells that received no treatment (NT). This demonstrates that neutron capture by [\u003csup\u003e10\u003c/sup\u003eB]-BPA increases the formation of DNA double-strand breaks and is significantly more pronounced than neutron irradiation alone. The pan-nuclear response observed at 10 min post-irradiation indicates that the damage is extensive and will likely result in cell death.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/c42506f603f6c05acc6253fc.jpg"},{"id":75405432,"identity":"afc6b91e-077c-4be2-8bba-4157f7bb26f4","added_by":"auto","created_at":"2025-02-04 08:47:13","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":53057,"visible":true,"origin":"","legend":"\u003cp\u003eγH2AX staining flow cytometric analysis of T98G cells gated on live DAPI\u003csup\u003e-\u003c/sup\u003e cells 60 min post neutron irradiation in the presence or absence of 500 μM [\u003csup\u003e10\u003c/sup\u003eB]-BPA. γH2AX staining intensity reveals a minor subset of cells with staining without neutron exposure with no detectable difference with or without BPA treatment. An increased exposure time to neutron irradiation correlates with an increased proportion of cells with increased γH2AXstaining intensity, with a majority of cells becoming highly positive for γH2AX following 10 min of neutron exposure when BPA is present.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/f7121db806d42c6eecfb885d.jpg"},{"id":83460670,"identity":"1401a958-ae91-4ae6-af43-b5c308fdb0ed","added_by":"auto","created_at":"2025-05-26 16:13:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":907355,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5800297/v1/b98c597f-5e14-4f22-8a63-393ad2004bca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adaptation of a neutron radiography instrument for live cell irradiation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeutron capture therapy (NCT) for the treatment of cancer is experiencing a resurgence of interest. This interest is being largely led by advances in compact accelerator-based neutron sources and appropriate beam shaping assemblies, decoupling this technique from expensive and unwieldy fission based sources, as well as advances in treatment planning software and patient positioning devices\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Broadly, NCT is an external beam radiotherapy modality that utilises neutron capture agents (NCA) - biochemically targeted stable isotopes of high neutron capture cross-section. Current methods approved for clinical use deliver \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB isotope to cancerous tissue via metabolic accumulation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Thermal neutron capture reaction in \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) is the foundational principle of boron neutron capture therapy (BNCT) and results in the production of a high linear energy transfer (LET) \u0026alpha;-particle (175 keV/\u0026micro;m), recoil \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003eLi nucleus (150 keV/\u0026micro;m) and a prompt 478 keV \u0026gamma;-ray, via the following reaction\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e:\u003c/p\u003e\n\u003cp\u003eThese particles\u0026apos; short path length offer a theoretically very high targeting specificity, causing lethal damage to the tumour or other targeted cells while sparing surrounding healthy tissue.\u003c/p\u003e\n\u003cp\u003eIn Japan, NCT has been approved for treating recurrent head and neck cancers, and promising results have been observed in other cancers like melanoma and brain tumours \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This body of evidence, together with the technical advancements incorporating NCT with particle therapy, presents a strong case for further investigation, and a wider adoption, of NCT principles for the treatment of cancer.\u003c/p\u003e\n\u003cp\u003eAdditionally, recent work has demonstrated that biologically relevant and clinically useful neutron fluxes are generated internally during proton and heavy-ion particle therapy, as a by-product of the particle irradiation itself \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Referred to as Neutron Capture Enhanced Particle Therapy (NCEPT)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, this technology extends the principles of NCT to applications in particle therapy (proton and carbon ion) treatments. Exploiting this thermal neutron field that is internally generated within the patient could potentially lead to significant reductions in primary ion beam dose and associated fractions, that are expected to directly benefit patient outcomes and quality of life as well as provide treatment options for some poor prognosis cancers.\u003c/p\u003e\n\u003cp\u003eIn order to optimise and progress NCT research, access to thermal neutron radiation sources are essential. In general, the rate limiting step for most radiotherapy basic research and development is accessing appropriate radiation sources. Access to the limited beam time available for research on clinical instruments is highly competitive and the costs associated with running these machines greatly limits the total time available. A low cost, simple, neutron irradiation capability, that has minimal impact on standard operations, allows for the efficient, iterative experimentation required for novel pharmaceutical development and analysis of radiobiological mechanisms. This manuscript describes the repurposing of a pre-existing neutron imaging instrument (referred to as \u003cem\u003eDingo\u003c/em\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, in use at the Open-Pool Australian Lightwater (OPAL) research nuclear reactor run by the Australian Nuclear Science and Technology Organisation, for the purposes of radiobiological research and novel NCT development. Dingo is currently the only thermal neutron beam available for research in Australia.\u003c/p\u003e\n\u003cp\u003eThe Dingo instrument is designed as a thermal neutron imaging instrument and its primary uses include palaeontology, materials science, archeology and industrial/engineering applications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. It is capable of neutron radiography and tomography and has two primary configurations:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eHigh resolution mode: 1 cm diameter pinhole producing a uniform field of 1.15x10\u003csup\u003e7\u003c/sup\u003en/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s at the sample stage\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/li\u003e\n \u003cli\u003eHigh intensity mode: 2 cm diameter pinhole producing 4.7x10\u003csup\u003e7\u003c/sup\u003en/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s at the sample stage\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eEven when operating in high-intensity mode the neutron flux at the sample stage is ∽10 lower than the minimum recommendation of 5x10\u003csup\u003e8\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s for reactor based BNCT \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The sample stage used for imaging sits 9.9 m from the primary shutter (Fig.\u0026nbsp;2). The inverse square law would indicate that halving the distance between the primary shutter and the sample could achieve a 4x increase in flux on the target, potentially increasing the flux to ∽2x10\u003csup\u003e8\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s, with further gains possible the closer to the primary shutter the sample can be located \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. These increases, while still lower than the minimum recommended for BNCT, put the flux within a range that is usable for \u003cem\u003ein vitro\u003c/em\u003e radiobiological and NCA structure-activity studies.\u003c/p\u003e\n\u003cp\u003eAn accessible location, immediately behind the tertiary shutter, was identified as a viable irradiation location. This positions the samples ∽4.8m from the primary shutter, effectively halving the distance, potentially generating the required increase in flux. As no sample stage is present at that location a mechanism to move the sample up to the tertiary shutter has to be developed. This system needs to efficiently and reproducibly position viable cell cultures at this location, which is at the entrance of the beam path to the instrument hutch on the opposite side of the 0.5 m thick concrete shielding wall. The dimensions of the tunnel through this wall, which sits in between the two helium flight tubes, is approximately 150 mm wide and 300 mm high.\u003c/p\u003e\n\u003cp\u003eWhen repurposing existing infrastructure for radiobiology, successful live cell irradiation has three major requirements:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eThe beam must be reliably located and its shape determined to allow effective targeting.\u003c/li\u003e\n \u003cli\u003eThe thermal neutron flux must be calculated so irradiation times can be determined and the fluence on the sample recorded.\u003c/li\u003e\n \u003cli\u003eCells must be placed into the path of the beam, while maintaining sterile conditions and viability, in a format suitable for the desired downstream analysis, for the correct period of time.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eIn addition, this work builds off the successful development of a fully validated Geant4 Monte Carlo simulation model of the Dingo beamline \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This \u0026ldquo;digital twin\u0026rdquo; simulates the entire beampath and allows precise prediction of the neutron field. This model is essential for ensuring the efficient use of the beam for irradiation purposes. This model has been validated in-beam and out-of-beam using a combination of absolute and relative measurements, including neutron activation analysis, Bonner sphere spectroscopy and a real-time quad-MOSFET device\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e which are now able to be employed for quality assurance and validation purposes during radiobiological experimentation. The primary technique used for neutron flux measurements on the beamline is NAA of high-purity bare and cadmium covered gold foils. Gold is sensitive to thermal and also epithermal neutrons with high-magnitude peaks of 2.7x10\u003csup\u003e5\u003c/sup\u003e barn and 3.6x10\u003csup\u003e3\u003c/sup\u003e barn at 4.9 eV and 60.3 eV, respectively, and several few-hundred barn resonance peaks. Therefore, to measure the thermal neutron flux, gold is usually paired with a cadmium-covered foil or wire. Cadmium is a highly efficient thermal neutron absorber, and so its activity, or a reaction rate corresponding to the epithermal neutron capture, can be subtracted from the bare gold separating the thermal neutron flux \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis work demonstrates that simple, low cost repurposing of existing beamlines is a viable approach for establishing a basic neutron radiation biology capability and has resulted in the first thermal neutron source available for this kind of research in Australia.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSample positioning\u003c/p\u003e \u003cp\u003eThe OPAL reactor supplies 15 instruments with neutron beams. However, one of its key applications is manufacturing of radiopharmaceuticals and neutron transmutation doping (NTD) of silicon. It requires inserting assemblies into the reactor core, which change the neutron scattering properties and may alter the spatial distribution of the neutron beams entering the beam port. Halving the distance between the pinhole and the samples increases not only the neutron flux, but also the variability in neutron intensities across the field, which is particularly important when operating in the high-intensity mode. Therefore, it is necessary to develop a robust and quick method to evaluate the relative position and planar distribution of the beam at Dingo.\u003c/p\u003e \u003cp\u003eA simple solution for efficient estimation of the neutron field characteristics using EBT3 radiochromic films (Ashland) placed behind a 1.2 mm cadmium sheet was implemented. The cadmium sheet is wrapped in aluminium foil and the film affixed to the back via tape. This assembly is attached to the sample holder, placed into the relevant location and irradiated for approximately 10 min. Following irradiation, the cadmium sheet is removed leaving the film in place and left to self-develop for another 10 min to visualise the position of the beam relative to the sample holder. This enables the acquisition of an almost instant relative image of the thermal neutron field via the low energy γ-rays produced by neutron capture in cadmium, and ensures the samples are placed in the desired planar region of the field. Since the reaction rates in the polymer depend on the deposited energy, the \u0026ldquo;instant\u0026rdquo; optical signal drops with decreasing neutron flux incident on the cadmium sheet. Thus, to determine the semiquantitative profiles of the neutron beam intensity, the film can be removed and self-developed for ~\u0026thinsp;12h to account for the non-uniform response. Finally, the film is scanned using a flatbed scanner to generate a 2D image. The optical density of the film is calculated from the pixel intensity which in turn is proportional to thermal neutron flux and can be plotted in ImageJ\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThermal neutron flux\u003c/p\u003e \u003cp\u003eThermal neutron fluxes were quantified by gold neutron activation technique using the methodology covered in Jakubowski \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The gold wires were pre-weighed and positioned on 2 cm x 2 cm cassettes containing the biological samples that were arranged in a 3 x 3 grid. Samples were then aligned to the geometric centre of the beam using the aforementioned technique and irradiated for 8h. The characteristic γ-ray emission of \u003csup\u003e198\u003c/sup\u003eAu was measured with a high-purity Germanium detector (HPGe). In this work, we used bare gold wires adjacent to the samples and the average thermal to epithermal neutron ratios to determine the thermal neutron fluxes. These were previously measured with cadmium-paired gold foils distributed across the field in the same location at Dingo\u0026rsquo;s tertiary shutter \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCell culture and Irradiation\u003c/p\u003e \u003cp\u003eU87-MG (CellBank Australia, 89081402) and T98G (CellBank Australia, 92090213) cells were propagated in Minimum Essential Media (MEM, Gibco 11095098), supplemented with 10%FBS, 100U/ml penicillin and 100\u0026micro;g/ml streptomycin, at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The day prior to irradiation 1x10\u003csup\u003e6\u003c/sup\u003e cells were seeded into a 24 well, optical base, tissue culture treated, assay plate to achieve\u0026thinsp;~\u0026thinsp;80% confluence. Cultures were treated with 500uM [\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA (\u0026gt;\u0026thinsp;98.4% \u003csup\u003e10\u003c/sup\u003eB, Interpharma Praha, Prague, Czech Republic), which was dissolved into cell media over a 2 hour time period at ~\u0026thinsp;40\u0026deg;C in an ultrasonic water bath, and left to incubate for 2 hours. All the wells were filled with warm D-PBS to reduce headspace and then sealed with a sterile adhesive film. The sealed plates were placed in the vertical position on the sample holder mounted to a specially designed trolley (Fig.\u0026nbsp;3A). The sample was manoeuvred into the irradiation position (Fig.\u0026nbsp;3B) and irradiated for a period of 1 min or 10 min, achieving an estimated fluence of 1.5x10\u003csup\u003e10\u003c/sup\u003en/cm\u003csup\u003e2\u003c/sup\u003e and 1.5x10\u003csup\u003e11\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e respectively.\u003c/p\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003cp\u003eFollowing irradiation, cells were incubated for 40 mins at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e after which they were fixed in 10% NBF and stored at 4\u0026deg;C in PBS until processing. The U87-MG and T98G cells were washed with PBS, and permeabilized with 0.1% Triton-X for 10 minutes at room temperature on a rocker. Samples were blocked with 5% BSA for 1h at room temperature on a rocker. Cells were then incubated with primary antibody (γH2AX anti-mouse (Merk Life Science, Australia) 1:1200 dilution in 1% BSA) overnight at 4\u0026deg;C, washed with 0.1% PBST, and incubated with secondary antibody (Alexa Fluor 488 goat anti-mouse (AbCam, Australia) 1:800 dilution in 1% BSA) for 1h at room temperature on a rocker. After a final PBST wash, cells were first counterstained with Phalloidin iFluor-594(1:1000, Abcam, Australia) for 1h and DAPI (4\u0026prime;,6-diamidino-2-phenylindole) (1 \u0026micro;g/ml) for 20 min at room temperature, washed again, and mounted using FluoroShield mounting media (Sigma). Samples were left to cure overnight at room temperature (protected from light) and stored at 4\u0026deg;C for up to two weeks prior to analysis.\u003c/p\u003e \u003cp\u003eThe resulting γH2AX foci were quantified by counting the number of foci per nucleus in 55 randomly selected cells, by eye, using a 100x objective. Images of foci were also taken using a 63x objective. Focal stacks of representative regions were acquired using a Zeiss Imager Z2 microscope and deconvolved using the Zen deconvolution package. Maximum intensity projections were prepared from the deconvolved stacks to illustrate the distribution of foci within the nucleus.\u003c/p\u003e \u003cp\u003eFlow cytometry\u003c/p\u003e \u003cp\u003eAt 60 min post irradiation, cells were washed with PBS and incubated in non-enzymatic cell dissociation solution (Sigma #C5914) for 5 to 10 min and kept on ice.\u003c/p\u003e \u003cp\u003eRecovered cells were washed and stained with DAPI (0.1\u0026micro;g/ml) followed by an additional 2 PBS washes. Cells were then permeabilized and stained with γH2AX (Phospho-Histone H2A.X (Ser139) (CR55T33), PE, eBioscience\u0026trade;) antibody using the BD (Becton-Dickinson) Pharmingen\u0026trade; Transcription Factor Buffer Set (#562574) following manufacturers\u0026rsquo; recommendations. Acquisition was performed on a 5 lasers BD FACSymphony\u0026trade; A3 Cell Analyzer. A minimum of 10000 DAPI\u003csup\u003eneg\u003c/sup\u003e cells were acquired per sample. Analysis was carried out using FlowJo\u003csup\u003e\u0026reg;\u003c/sup\u003e software.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Mann-Whitney U test was employed to analyse differences between groups in visualised γH2AX foci data. Visual analysis of the data showed foci per nucleus demonstrated a potentially skewed nature within this cell population. This skewing is due to the high background of spontaneous foci formation often present in cancer cells. This test was used as it compares medians and is sensitive to distribution differences, therefore able to provide insights into how these treatments differ.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAs previously demonstrated by Jakubowski et al., the neutron beam at Dingo constitutes approximately 59% thermal, 21% epithermal and 20% fast neutrons\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Imaging of the beam with radiochromic films shows an approximate square shape (Fig. 3). The x axis shows a relatively flat profile with sharp shoulders and fluxes slightly skewed to the left. The y axis profile has rounded shoulders and a more pronounced drop off in thermal neutron flux towards the top of the beam. The relatively flat area available for sample irradiation at the irradiation position is 10 cm in the x axis and 6 cm in the y axis with the distance in y being limited by the rounded shoulders. Location in the tunnel is determined relative to the sample holder, allowing the samples to be reliably and reproducibly placed into this location.\u003c/p\u003e\n\u003cp\u003eThe relative thermal neutron spatial distribution shows good agreement with the results of the absolute thermal neutron flux measurements using gold wires (Fig.\u0026nbsp;4). The 8 x 8 cm array, positioned in the centre of the beam, measured a neutron flux of 2.57x10\u003csup\u003e8\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s (\u0026plusmn;\u0026thinsp;2.73x10\u003csup\u003e7\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eThe number of \u0026gamma;H2AX foci per nucleus increased significantly with the addition of 500\u0026micro;M [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA when compared to irradiation alone demonstrating the presence of a neutron capture dose (Fig.\u0026nbsp;5). A Mann-Whitney U test was implemented to compare foci/nucleus between the 10min irradiation groups with and without the addition of [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA. There was a significant difference (U\u0026thinsp;=\u0026thinsp;867.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) between the untreated (median\u0026thinsp;=\u0026thinsp;8.5 (LL\u0026thinsp;=\u0026thinsp;8.112, UL\u0026thinsp;=\u0026thinsp;10.23), n\u0026thinsp;=\u0026thinsp;55) and treated (median\u0026thinsp;=\u0026thinsp;12.0 (LL\u0026thinsp;=\u0026thinsp;11.38, UL\u0026thinsp;=\u0026thinsp;14.26), n\u0026thinsp;=\u0026thinsp;55) cells. A lower significance change (U\u0026thinsp;=\u0026thinsp;969.5, p\u0026thinsp;=\u0026thinsp;0.0010) was observed when comparing the 1 minute irradiation group (median\u0026thinsp;=\u0026thinsp;12.0 (LL\u0026thinsp;=\u0026thinsp;10.85, UL\u0026thinsp;=\u0026thinsp;14.02), n\u0026thinsp;=\u0026thinsp;55).\u003c/p\u003e\n\u003cp\u003eQuantification of foci in this manner was complicated in these cell lines by the presence of a very high background of foci production, which can often be seen in cancer cell lines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The background foci per nucleus of these cultures is around 10 and results in non-normally distributed data. This technique of counting foci also has limitations at higher doses with the foci becoming too close together to reliably distinguish one from the other leading to inaccuracy in counting.\u003c/p\u003e\n\u003cp\u003eDeconvolved z-stack images of these cultures demonstrate the nature of \u0026gamma;H2AX staining observed during the counting procedure (Fig.\u0026nbsp;6). A visual increase in both foci number and fluorescence intensity can be observed that correlates with the time of irradiation. Cells treated with 500\u0026micro;M [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA show increased levels of foci formation before tending to high levels of pan-nuclear staining following the 10 min irradiation.\u003c/p\u003e\n\u003cp\u003eThis increase in fluorescence intensity along with foci formation was further investigated by flow cytometry (Fig.\u0026nbsp;7). It showed an irradiation time dependent increase in fluorescence intensity in response to staining for \u0026gamma;H2AX, following incubation with 500 \u0026micro;M [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA. A minor subset of cells exhibiting \u0026gamma;H2AX staining is present in both [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA and NT controls that increases with irradiation time, becoming the majority of the population in the cultures treated with [\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB]-BPA following 10 mins of neutrons.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo the best of our knowledge, this is the first, and only, neutron beam available for radiation biology research in Australia. This work demonstrates that low cost, repurposing of existing beamlines is a viable approach for the establishment of a neutron radiation biology capability. No permanent alterations to the existing infrastructure were required to achieve the successful irradiation of viable human tissue cultures at biologically significant dose rates.\u003c/p\u003e \u003cp\u003eBy utilising commercially available radiochromic films, we employed a robust and almost instant method to evaluate potential changes to the relative position and spatial distribution characteristics of the thermal neutron component of the beam at Dingo, which can occur due to modifications of the reactor core configuration. This approach increases experimental reproducibility and improves the positional accuracy of the samples. The thermal neutron flux can be effectively measured using gold NAA technique.\u003c/p\u003e \u003cp\u003eMeasurements of the thermal neutron flux at the irradiation position are in good agreement with the previously published fully validated Monte Carlo model of the beam\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Reported thermal neutron flux when running in high intensity mode at the sample stage is 4.7x10\u003csup\u003e7\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s. It accounts for approximately 59% of the neutron beam while the epithermal and fast neutron components comprise 21% and 20%, respectively. By approximately halving the distance to the primary shutter, the thermal neutron flux increases by nearly the factor of 4. It can be further increased by placing the samples immediately behind the tertiary shutter entry reaching 2.52x10\u003csup\u003e8\u003c/sup\u003e (\u0026plusmn;\u0026thinsp;2.73x10\u003csup\u003e7\u003c/sup\u003e) n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s. A previous experimental irradiation of tissue cultures at the Dingo sample stage used the 4.7x10\u003csup\u003e7\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s flux to deliver 1.2x10\u003csup\u003e12\u003c/sup\u003en/cm\u003csup\u003e2\u003c/sup\u003e over a 7h time period\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Cells were kept in zero headspace at room temperature for the duration, which can introduce major confounding factors to the analysis of cell survival and proliferation. Repeating this experiment with methodology described above would result in a reduction of irradiation times to ~\u0026thinsp;1.3h, with further reductions possible if the irradiation is restricted to the highest flux portion of the beam.\u003c/p\u003e \u003cp\u003eViable human tissue cultures placed behind the tertiary shutter and irradiated for periods of up to 10 min; the cultures were successfully recovered and the resulting activation of HR and NHEJ DNA repair pathways measured by γH2AX immunocytochemistry and flow cytometry. Significant increases in DNA damage were observed from the inclusion of 500\u0026micro;M \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003eB-BPA, demonstrating the successful application of thermal neutron radiation to these samples.\u003c/p\u003e \u003cp\u003eThere are two major limitations that must be taken into consideration when irradiating tissue cultures as described above:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe horizontal geometry of the beam requires that tissue cultures be held in a vertical position and;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIrradiations are conducted under ambient conditions.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eTo address point 1 all irradiations occur within air tight sealed containers, such as a 24 well plate with an adhesive seal or a screw top flask. To prevent cultures dehydrating during setup and irradiation time all containers are prepared with as close to zero headspace as practically achievable. In order to reduce the impacts of both points it is important to minimise the time the cultures are exposed to these conditions. Efficient sample setup and retrieval is key to keeping the impacts of these factors consistent between irradiations. The significance of these factors needs to be taken into consideration on a per cell line basis for future work.\u003c/p\u003e \u003cp\u003eInclusion of temperature control to the irradiation setup would be a valuable addition to this technique as it would allow for maintaining conditions in the cell cultures that are closer to physiological. Compact heating devices, such as Peltier heat exchanges that can be thermostatically controlled are being investigated. The complexities of placing such devices into such a neutron rich environment need to be taken into consideration from a functional and longevity perspective as well as the potential activation of the components. The current irradiation setup is constructed entirely out of plastic which does not activate to any significant extent under these conditions.\u003c/p\u003e \u003cp\u003eThe gold neutron activation analysis technique used in this study is the \u0026lsquo;gold standard\u0026rsquo; for calculating thermal neutron flux coming from nuclear reactors. As this technique directly measures the activation of \u003csup\u003e198\u003c/sup\u003eAu via gamma ray spectrometry, the quantification of the thermal neutron flux is straightforward and robust. The technique itself is time-consuming compared to the short irradiation times of 1 min or less used for the irradiation of biological samples, with results being received a minimum of 24 h post experiment. It also requires sufficient gold activation for reliable quantification of the gamma ray emission and calculation of thermal neutron fluxes. The addition of a real-time neutron monitoring device that can distinguish between thermal and epithermal neutrons would be a very valuable addition. A device using a combination of MOSFET detectors covered with either boron carbide, cadmium and boron carbide, or polyethylene converters has been tested in simulation with very promising results\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Since the neutron beam properties can vary between the measurements, real-time feedback will be extremely valuable to allow optimisation of experimental protocols to more efficiently utilise the available beamtime and improve both the precision, and accuracy of irradiations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe primary rate limiting factor for NCT research and development is access to appropriate radiation sources. Typically, hours on such instruments are limited and the merit based access models extremely competitive. To accelerate development of novel NCAs and targets, more beamtime is required. We have developed the techniques necessary to repurpose Dingo, a thermal neutron imaging instrument the OPAL reactor in Australia, to conduct biological irradiations for the purposes of radiobiological research and development. Sample positioning and techniques for robust beam characteristics evaluation and neutron flux measurements were developed to provide a radiobiological irradiation capability with no major changes to existing infrastructure. Opportunities for further development to improve precision and accuracy have been identified. It is hoped that by encouraging others to explore the potential of existing nuclear reactor-based facilities for biological research we can accelerate development in the field in order to meet the demand, and take full advantage, of the lower cost, modern accelerator-based neutron sources and techniques to utilise internally generated neutrons - a by-product of particle therapy treatment that will be available in the near future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.H, F.S and R.J. conducted all biological experiments.C.D synthesised and provided all chemical compounds and assisted with methodological development.J.B ad U.G provided technical support, and access for all beamline operations.K.J and M.S provided the Monte Carlo model and support for physics measurements.N.H, F.S and K.J prepared the manuscript.The manuscript was reviewed by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAtila Stopic - gamma ray spectroscopy analysis of neutron activated gold.Deborah Wakeham - assistance with process development and logistics.Luca Daveos - assistance with design and engineering of sample holder.Australian Centre for Neutron Scattering (ACNS) - for availability of Dingo via access grants P16726, 17623 and 18678, and for supplying regular beamtime for work that falls well outside the scope of regular operations.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated and analysed in this publication is available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoron neutron capture therapy. 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(2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirzayans, R. et al. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 11609\u0026ndash;11628 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, W. et al. Cyclic-RGDyC functionalized liposomes for dual-targeting of tumor vasculature and cancer cells in glioblastoma: An in vitro boron neutron capture therapy study. \u003cem\u003eOncotarget\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 36614\u0026ndash;36627 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5800297/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5800297/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeutron Capture Therapy (NCT) for cancer treatment is experiencing renewed interest due to advancements in accelerator-based neutron beams, treatment planning software, and patient positioning devices. This study presents the repurposing of an existing neutron radiography beamline (Dingo), at the OPAL research nuclear reactor, for radiobiological research and novel NCT development. Human glioblastoma cell cultures were irradiated for up to 10 mins with a flux of 2.57x10\u003csup\u003e8\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e\u0026sdot;s (\u0026plusmn;\u0026thinsp;2.73x10\u003csup\u003e7\u003c/sup\u003e) and the resulting impact was quantified by assessing DNA damage by both immunocytochemistry and flow cytometry. This low cost methodology extends the capability of an existing beamline to allow the development of novel neutron capture agents and study of neutron radiobiological mechanisms. Increasing availability of neutron sources for biological study in this fashion will accelerate the development of NCT for disease specific clinical application.\u003c/p\u003e","manuscriptTitle":"Adaptation of a neutron radiography instrument for live cell irradiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-04 08:47:07","doi":"10.21203/rs.3.rs-5800297/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-26T06:53:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-15T17:07:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-31T02:26:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309983862518107949699288346856977692687","date":"2025-01-25T19:30:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337837856126710292378843598719126105666","date":"2025-01-21T10:24:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-20T19:23:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-20T19:18:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-18T05:54:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-17T03:28:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-10T03:41:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ad025be0-ffa4-481b-a889-a8464a83c93b","owner":[],"postedDate":"February 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43649769,"name":"Biological sciences/Cancer"},{"id":43649770,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":43649771,"name":"Biological sciences/Cancer/Cancer therapy/Radiotherapy"},{"id":43649772,"name":"Biological sciences/Drug discovery/Drug screening"},{"id":43649773,"name":"Biological sciences/Cell biology/Cell death"},{"id":43649774,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Radiotherapy"}],"tags":[],"updatedAt":"2025-05-26T16:10:20+00:00","versionOfRecord":{"articleIdentity":"rs-5800297","link":"https://doi.org/10.1038/s41598-025-02382-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-22 15:58:14","publishedOnDateReadable":"May 22nd, 2025"},"versionCreatedAt":"2025-02-04 08:47:07","video":"","vorDoi":"10.1038/s41598-025-02382-4","vorDoiUrl":"https://doi.org/10.1038/s41598-025-02382-4","workflowStages":[]},"version":"v1","identity":"rs-5800297","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5800297","identity":"rs-5800297","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}