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

该项目旨在通过将X射线生产和玻璃毛细管光学的最新发展与单细胞靶向和分析技术相结合，开发一种独特的紧凑型高能(5-25keV)X射线微束设备。该设施将是以前所未有的方式调查简单和复杂生物样本对一种在我们社会中广泛使用的电离辐射(从医学诊断和治疗应用到核和环境水平)的风险和反应的精致工具。精细的分辨率、宽的能量范围、亮度和紧凑的尺寸将使该设备独一无二，不仅适用于放射生物应用。这一目标将通过改进商业上可用的X射线源和采用玻璃毛细管装置将X射线聚焦到微米和亚微米直径的斑点来实现。光源的改进将主要针对增加光源亮度(即X射线生产)，同时减少有效X射线光源(10微米直径)。目标冷却选项(钻石散热器和Peltier单元)也将被考虑以增加输出通量，从而产生点状、非常明亮的实验室工作台X射线源。通过利用浅入射角的全反射，玻璃毛细管设备将能够通过多次内部反射将硬X射线聚焦到一个细小的光点。具体地说，我们的目标是将~1GY/秒发射到亚微米级的光斑。最后，开发的硬X射线微焦点探测器将被集成到现有的单细胞照射设施中。这种系统由3轴微定位工作台(0.25微米分辨率)连接到表观荧光显微镜，并由内部开发的软件控制，以自动定位生物细胞和亚细胞目标，并将它们与特定的辐射探针对准。放射生物微束能够将特定剂量的辐射传递到单个或部分细胞，然后评估所造成的损害和所造成的影响。因此，微束是精确研究辐射对生物样本的影响以及调节细胞对辐射伤害反应的复杂途径的独特工具。尽管确定性辐照实验的重要性自20世纪50年代初被认识到，但直到最近几十年的技术进步，才有可能发展出精密的微束。在此期间，微束对我们在辐射生物学方面的知识做出了重大贡献，提供了已经并正在被用于放射治疗和辐射防护目的的关键见解。目前，世界上大多数微束设备使用带电粒子，只有3个使用软X射线(&lt；5kev)。另一方面，硬X射线(&gt；5kev)因其衰减特性、电离/损伤模式以及在现代社会中的广泛使用(从诊断设备到自然和人为本底水平)而特别令人感兴趣。硬X射线微束将用于广泛的放射生物学实验，旨在研究与极低剂量稀疏电离辐射相关的影响和风险。特别是，靶向选定群体或实际上复杂的3D组织结构内的单个细胞的能力将为研究旁观者效应(即，在不直接暴露的细胞中表达的辐射效应，但在接触或接近辐照样品时表现出来)提供宝贵的资产。此外，亚核细胞器(即线粒体)和单个染色体可以作为靶标，以研究它们的辐射抗性并解决有关其功能的具体问题。最后，我们在开发高能X射线微焦点方面的发现和专业知识也可能对X射线显微镜和光谱学领域有利。

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.