Optical Imaging of Uranium Biotransformations by Microorganisms (OPTIUM)

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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.