Development of a hard X-ray microfocus source for radiobiological applications

The project aims to develop a unique compact high energy (5-25 keV) X-ray microbeam facility by integrating recent developments in X-ray production and glass capillary optics with single cell targeting and analysis technique. The facility will represent an exquisite tool to investigate risks and responses of simple and complex biological samples to a type of ionizing radiation widely used in our society (from medical diagnostic and therapeutic applications to nuclear and enviromental levels) in an unprecedented way. The fine resolution, wide energy range, brightness and compact size will make this facility unique and appealing not just for radiobiological applications. Such a goal will be realised by improving commercially available X-ray sources and adopting glass capillary devices to focus X-rays into micron and submicron diameter spots. Source improvements will be mainly directed to increasing the source brightness (i.e. X-ray production) while reducing the effective X-ray source (< 10 micron diameter). Target cooling options (diamond heat spreaders and Peltier units) will also be considered to increase the output flux and producing therefore a point-like, very bright lab bench X-ray source. By exploiting the total reflection that occurs at shallow incident angles, glass capillary devices will then able to focus hard X-rays into a fine spot through multiple internal reflections. Specifically, we aim to deliver ~1 Gy/sec into sub-micron spots. Finally, the developed hard X-ray microfocus probe will be integrated into an existing single-cell irradiation facility. Such a system consists of a 3-axis micropositioning stage (0.25 micron resolution) coupled to an epi-fluorescent microscope and controlled by in house developed software to automatically locate biological cellular and sub-cellular targets and align them with a specific radiation probe.Radiobiological microbeams are facilities able to deliver a specific dose of radiation to single cells or part of them and subsequently assess the damage induced and the effect caused. As such, microbeams are unique tools to precisely investigate effects of radiation on biological samples and the complex pathways that regulate cellular response to radiation insult. Despite the importance of a deterministic irradiation experiment has been recognised since the early 1950's, only with the technological advances of the last couple of decades has it been possible to develop sophisticated microbeam. Over such a period, microbeam have significantly contributed to our knowledge in radiation biology providing critical insights which have and are being exploited for radiotherapy and radioprotection purposes. Currently most of the microbeam facilities worldwide use charged particles and only 3 employ soft X-rays (<5 keV). On the other hand, hard X-rays (>5 keV) are particularly interesting due to their attenuation characteristics, the pattern of ionization/damage induced and their wide use in modern society (from diagnostic equipment to natural and man-made background levels).The hard X-ray microbeam will be used for wide range of radiobiological experiments aimed to study the effects and risks associated with exposure to very low doses of sparsely ionizing radiation. In particular, the ability to target individual cells within a selected populations or indeed a complex 3D tissue structure will provide a valuable asset for the investigation of the bystander effect (i.e. radiation effects expressed in cells not being directly exposed but in contact or proximity of irradiated samples). Moreover, sub-nuclear organelles (i.e. mitochondria) and individual chromosomes can be targeted in order to investigate their radioresistance and address specific questions about their functionality. Finally, our findings and expertise in developing high energy X-ray microfocus could also be beneficial to the X-ray microscopy and spectroscopy communities.

The proposed multidisciplinary project will impact on a range of research areas with potential benefits ranging from radiobiology to X-ray optics and radiation physics for individuals, academic and national research facilities as well as commercial organizations. Such benefits and plans to realise them are discussed below. The hard X-ray microbeam is expected to greatly contribute to advances in the radiobiological field. The great targeting accuracy (sub-micrometer radiation probe) and experimental flexibility (from single cells to 3D biological models) will make it possible to investigate the effect of low LET radiation in an unprecedented manner. Through publications in high impact journals and conferences presentations/proceedings, project progress and experimental data will be divulgated to the radiobiology, radiation oncology and radioprotection community with the intent to provide expert insights to improve current applications and regulations dealing with use or exposure to high energy X-rays (i.e. medical diagnostic and therapeutic applications, nuclear and natural background). The hard X-ray microbeam facility will be available to scientists of the CCRCB (Queen's University Belfast) and external users through new and existing collaborations. The project will also contribute to improve the scientific profile of use of microirradiation facilities in radiobiology and more generally the multidisciplinary approach promoted by CCRCB. The new hard X-ray microbeam will represent a landmark for radiobiological microbeams as the use of bench-top, commercially available radiation source and glass capillary optic will result in a compact facility which could be implemented and run in a conventional biological laboratory without the need of expensive and cumbersome equipment and dedicated technical assistance. Our expertise in automated micropositioning, sub-cellular targeting and first-generation microbeam development will be crucial to provide proof of principle and development details to empower the radiobiological community. The proposed research is also expected to impact the field of X-ray optics as the use of glass capillary systems to focus high energy X-rays will further be investigated. Despite their relatively straightforward use, fine resolution, energy independence and mature manufacturing technology, glass capillaries have so far been poorly exploited. With input from world experts in X-ray optics, the proposal will investigate practical applications of glass capillaries contributing to developing their applications and potential. A direct comparison with alternative innovative approaches will also be performed for the direct benefits of collaborators. Data generated is expected to be of interest for the X-ray microscopy and spectroscopy community. The successful development of a bench-top hard X-ray microbeam can also be considered as the first step leading to a more powerful and versatile facility based at Diamond Light Source Synchrotron. Skills and expertise gained for the proposed project will be used to adapt the glass capillary technology to synchrotron light sources. On the other hand, our existing micropositioning, image analysis and sub-micrometer targeting expertise will be crucial for the development of a suitable end station to fully exploit the fine X-ray beam produced. A nanobeam targeting facility which will take advantage of Diamond Light Source high photon flux and wide range of monochromatic X-rays, will represent a valuable asset for a synchrotron beamline and its users and a powerful tool to perform not just radiobiological experiments. Finally, the development of a unique microirradiation facility will strengthen the role of Dr. Schettino and UK collaborators as the world leaders in this field.