A Facility for Advanced Imaging and Analysis under Hypoxic Conditions for Bath Scientists and GW4 partners

We wish to develop a unique and cutting edge facility in Bath to enable us to study the impact that low oxygen conditions (hypoxia) have on cell function. Oxygen is vital for life and inspired from air. In a healthy individual, oxygen levels in the circulation are lower than atmospheric and decrease further depending on how far from the blood supply. For example some brain tissue and skin layers are a significant distance from a blood supply. In the normal situation, oxygen levels in most tissues would be considered low (hypoxic) compared to atmospheric oxygen levels. At sites of inflammation the level of oxygen decreases further due to increased oxygen usage by cells and a reduced blood supply. These changes affect the function of many tissues including the brain which is particularly susceptible to hypoxia resulting from transient ischemic episodes or impaired blood flow during aging. The level of oxygen dramatically alters many cell properties, including the genes expressed and protein function. In modern science it is essential to derive accurate models of cell function in the lab so results can be translated to the living system. In many lab-based experiments, isolated cells are cultured in atmospheric oxygen levels that do not reflect the environment encountered in the healthy body. The 'advanced hypoxic imaging facility' will use high performance imaging and analytical equipment with user defined oxygen levels to study different aspects of cell function. This state of the art approach represents a significant technical advance that will enable us to very accurately mimic the hypoxic conditions that occur in humans in our cellular model systems. The instruments will measure: (1) activity of single cells with an ultrasensitive, high speed detection camera; (2) multiple conditions using a high-throughput reader with a revolutionary technology for increased detection sensitivity; and (3) modified oxygen species using 'electron spin resonance'. The proposal includes five specific projects to measure the properties of immune cells found at the sites of inflammation, brain cells and cells that form blood vessels. The instruments will form part to the Bio-imaging Suite in the Microscopy and Analysis Suite in the University of Bath. The user community will include scientists in the South West and South Wales.

We aim to establish a world-leading, high-sensitivity, high-resolution, imaging and analytical facility to study cell function under hypoxic conditions. In 21st century bioscience it is vital to build accurate in vitro models of cell signalling that translate to the living system. To achieve this objective the 'Advanced Hypoxic Imaging Facility' will bring together three cutting edge instruments to provide a pioneering approach to study in vitro cell function. Each instrument will measure different aspects of cell or molecular function under continuous hypoxic conditions that collectively will form a world-class facility. We will use the following advanced methodologies: (1) A Ruskinn Sci-tive dual asymmetrical hypoxic workstation with two independent chambers with individually controlled atmospheres connected by an airlock. The second chamber will accommodate a Leica DMI6000B AFC inverted microscope with an ultrasensitive Andor iXon Ultra 897 EMCCD camera. This is a pioneering combination of equipment where there will be continuous hypoxic conditions from cell culture in chamber one to high-resolution spatiotemporal imaging in the second chamber. (2) A revolutionary, high sensitivity CLARIOstar microplate reader with an atmospheric control unit to regulate oxygen and carbon dioxide levels. (3) Bruker Bio-III e-scan system has regulated temperature and gas control to facilitate the measurement of reactive oxygen and nitrogen species under hypoxic conditions using electron paramagnetic resonance. These high performance instruments will provide advanced imaging and analytical measurements from high throughput screening, quantification of reactive oxygen species and ultra-sensitive, high-speed fluorescent imaging under hypoxic conditions. The applicants will use the facility to probe fast receptor trafficking, cell motility, protein-protein interaction kinetics, synaptic dysfunction and redox imbalance, mitochondrial and lysosomal function.

This advanced research technology with the pioneering use of environmental gas control will strongly impact on the competitiveness of the UK research base and contribute to the UK knowledge economy. This proposal strongly aligns with the 'BBSRC research priority 3' to drive advances in bioscience for better health across the life course. This priority aims to achieve an integrated understanding of the healthy system including maintaining health under stress or environmental challenge including the aging process (associated with increased hypoxia) plus effects on the immune system and the brain. This bid will use bioimaging, analytical and mathematical approaches to generate new knowledge of biological systems that will impact on our understanding of the healthy system.

This work will have significant impact on a) the academic community by providing better models of cell signaling under physiological conditions; b) skills training of postdoctoral and postgraduate researchers that will enhance graduate employability within academia and industry; c) the links between researchers and the private sector including charities (e.g. Alzheimer's Society, CRUK, BHF) and pharmaceutical companies (e.g. Janssen Pharmaceutica, Novartis, Medimmune); d) GW4 consortium by increasing research infrastructure and promoting collaborative research and e) cross-disciplinary research including mathematical biology and policy research.

Replacement, reduction and refinement (3Rs) of animals in research is a priority in the UK that led to the establishment of the National Centre for the 3Rs of animals in research (NC3Rs) in 2004. The advanced hypoxic imaging facility will impact on UK and international policy makers by providing better in vitro methodologies to generate models of cell function. Understanding the properties of cells under hypoxic conditions will be a powerful approach to provide accurate in vitro models to inform and ultimately reduce animal experiments; this is strongly aligned with BBSRC's commitment to the 3Rs approach to animal research. As such, the Hypoxic Imaging Facility will also be of significant public interest as this addresses public concerns regarding the use of animals in research. The BBSRC ALERT14 bid will be announced to the public and research outputs disseminated through a range of public engagement activities.