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（5-25 keV）X射线微束设施。该设施将代表一种精致的工具，以前所未有的方式调查简单和复杂生物样品对我们社会广泛使用的一种电离辐射的风险和反应（从医疗诊断和治疗应用到核和环境水平）。精细的分辨率，宽的能量范围，亮度和紧凑的尺寸将使该设施不仅在放射生物学应用中具有独特的吸引力。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射线聚焦成微米和亚微米直径的斑点来实现。源的改进将主要针对增加源亮度（即X射线产生），同时减少有效X射线源（直径< 10微米）。还将考虑目标冷却选项（金刚石散热器和Peltier装置），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。通过利用在浅入射角下发生的全反射，玻璃毛细管装置将能够通过多次内反射将硬X射线聚焦成精细的点。具体而言，我们的目标是将~1戈伊/sec的剂量传递到亚微米斑点中。最后，开发的硬X射线微焦点探针将被集成到现有的单细胞辐照设施。这种系统包括一个3轴微定位平台（0.25微米分辨率）耦合到落射荧光显微镜，并由内部开发的软件控制，以自动定位生物细胞和亚细胞，放射生物学微束是能够将特定剂量的辐射传递到单个细胞或其中的一部分，随后评估诱导的损伤，造成的影响。因此，微束是精确研究辐射对生物样品的影响以及调节细胞对辐射损伤的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年代初就已被认识到，但只有随着最近几十年的技术进步，才有可能开发出复杂的微束。在此期间，微束为我们的辐射生物学知识做出了重大贡献，提供了重要的见解，这些见解已经并正在被用于放射治疗和辐射防护目的。目前，世界上大多数微束设施使用带电粒子，只有3个使用软X射线（<5 keV）。另一方面，硬X射线（>5 keV）由于其衰减特性而特别令人感兴趣，电离/损伤模式及其在现代社会中的广泛应用（从诊断设备到自然和人为背景水平）。硬X-射线微束将用于广泛的放射生物学实验，旨在研究与暴露于极低剂量的稀疏电离辐射特别是，靶向选定群体内的单个细胞或实际上复杂的3D组织结构的能力将为研究旁观者效应（即，在未直接暴露但接触或接近辐照样品的细胞中表达的辐射效应）提供有价值的资产。此外，可以靶向亚核细胞器（即线粒体）和单个染色体，以研究其辐射抗性并解决有关其功能的特定问题。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社区有益。

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.