Optical Imaging of Uranium Biotransformations by Microorganisms (OPTIUM)

当今社会面临的最紧迫问题之一是对核能生产产生的现有和未来废物形式的管理。虽然放射性在环境中自然存在，但60多年的人为活动，包括采矿、工业核电生产、核材料的意外泄漏和军事使用，导致自然环境中的放射性核素水平大大增加。虽然在许多情况下，污染是集中的，并不广泛，但这些放射性核素对更广泛的生态系统造成的影响与所涉放射性核素的生物利用度错综复杂地联系在一起，这是由其浓度和化学形态(氧化态和形态)决定的。鉴于重金属铀在质量上构成了大部分废物，在管理安全处置以防止浸出方面，将铀从其可溶的、因此可移动的形式(铀酰(VI))化学转化为基本上不可溶的、因此不可移动的形式(铀(IV)矿物形式)是一项重要的战略。各种微生物过程通常涉及细菌介导的氧化还原转化，已被认为是可行的生物修复技术。通常，这些反应是通过X射线吸收技术、纯定量技术或电子显微镜在固定(死亡)细胞上进行研究的。目前缺乏能够定量探测放射性核素的分布和微环境的技术，特别是在活细胞中。在这里，我们建议引入强大的双光子荧光显微镜技术，利用铀酰(VI)离子的本征发射信号在体内跟踪和揭示这些微生物过程，以便在高空间分辨率下对所提出的原位生物修复过程有一个全面的了解。双光子显微镜目前在生物学中被广泛用于三维可视化细胞过程，但尚未被用于成像涉及铀的细胞过程。与紫外线/可见光激发相比，铀酰阳离子的基本光物理性质将使双光子激发在电磁光谱的近红外区具有较小的破坏性，而紫外/可见光激发在单光子过程中对细胞造成损害。长寿命的铀酰排放本身(参看。染料)和双光子激发的固有空间控制使含铀酰的生物材料能够高分辨率可视化，而荧光寿命图则展示了可视化U(VI)还原细菌细胞表面微观氧化还原条件的能力。首次使用无损3D多光子光学成像技术与最先进的光谱学相结合，将作为这一研究领域的一项新技术开发，并用作工具来解决在一系列生物地球化学系统(这里是细菌和真菌)中了解铀形态和反应性的挑战。我们的目标是利用铀离子的内在光学性质作为直接可见发射探针，因为它们与这些微生物在化学上相互作用，从而达到更具地质学意义的时间尺度。我们的总体愿景是实施3D光学成像，以以前未曾见过的细节水平(亚微米和亚毫微秒时间尺度)识别和成像铀离子及其形态，并通过X射线和电子显微镜方法来增强这一点，以创建一个新的工具箱，用于了解微生物和真菌系统，这些系统生物积累、生物转化和生物矿化放射性有毒和对环境有害的榄系元素离子，使其形成流动性较小的形式。通过与一系列关键利益攸关方(例如，放射性废物管理有限公司、国家核实验室)合作，我们可以使用这种光学成像技术更好地预测受污染地点的放射性核素迁移率，并为英国和更广泛地区的处置和土地管理提供信息。

One of the most pressing problems facing society today is the management of existing and future waste forms arising from nuclear energy production. Although radioactivity is naturally occurring in the environment, 60+ years of anthropogenic activities including mining, industrial nuclear power production, accidental release and military use of nuclear materials has led to greatly increased levels of radionuclides in the natural environment. Although, in many cases, the contamination is concentrated and not widespread, the impact of these radionuclides pose to the wider ecosystems is intricately linked to the bioavailability of the radionuclide in question, which is dictated by their concentration and chemical form (oxidation state and speciation). Given that the heavy metal uranium comprises the majority waste by mass, the chemical transformation of uranium from its water soluble, and therefore mobile form (uranyl(VI)) to essentially an insoluble, and therefore immobile form (uranium(IV) mineral forms) is an important strategy in managing safe disposal to prevent leaching. Various microbial processes, often involving bacterially mediated redox transformations, have been suggested as viable bioremediation techniques. Typically these reactions are studied on the bulk level by X-ray absorption techniques, using purely quantitative techniques or on fixed (dead) cells by electron microscopy. There is currently a lack of techniques that are capable of quantitatively probing the distribution and micro- environment of radionuclides, particularly in living cells. Here we propose to introduce the powerful technique of two-photon fluorescence microscopy using the intrinsic emissive signals of the uranyl(VI) cation to follow and unravel these microbial processes at the sub-micron level in vivo in order to gain a full understanding of the proposed bioremediation process in situ at high spatial resolution. Two-photon microscopy is currently widely used in biology to visualise cellular processes in three dimensions, but has not yet been used to image cellular processes that involve uranium. The fundamental photophysical properties of the uranyl cation will enable two-photon excitation in the less damaging near infra-red region of the electromagnetic spectrum compared to UV/visible excitation which is damaging to cells in a one photon process. The long-lived uranyl emission itself (cf. dyes) and inherent spatial control of two-photon excitation allow high-resolution visualisation of uranyl-containing biological material, while fluorescence lifetime mapping demonstrates the ability to visualise the microscopic redox conditions over the surface of U(VI)-reducing bacterial cells. The first ever use of non-destructive 3D multi-photon optical imaging techniques combined with state of the art spectroscopy will be developed as a new technology in this research field and used as tools to address the challenge of understanding uranium speciation and reactivity in a range of biogeochemical systems, here, bacteria and fungi. We aim to exploit the intrinsic optical properties of the uranium ions as direct visible emissive probes as they interact with these microorganisms on chemical to more geologically relevant timescales. Our overall vision is to implement 3D optical imaging to both identify and image uranium ions and their speciation at a previously unseen level of detail (sub micron and sub ns timescale) and augment this with X-ray and electron microscopy approaches to create a new toolbox for understanding microbial and fungal systems that bioaccumulate, biotransform and biomineralise radiotoxic and environmentally hazardous actinide ions into less mobile forms. Working with a range of key stakeholders (e.g. Radioactive Waste Management Ltd., National Nuclear Laboratory), we can use this optical imaging technique to better predict radionuclide mobility at contaminated sites and inform disposal and land management in the UK and wider afield.