MICA: Liverpool Imaging Partnership: Molecular physiology and drug response

In multicellular organisms, cells are exposed to molecular signals such as inflammatory molecules and stress signals. Cells have to interpret these signals to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g. cell death, cell growth, cell migration, etc), biologists have to measure the levels, localisation and modifications of proteins, and the subsequent regulation of specific genes. There is an increasing recognition that in a population of cells, each cell is different and therefore measuring the average behaviour can lead to erroneous conclusions. Live cell imaging coupled with the use of fluorescent and luminescent labels and reporters enables biologists to monitor dynamic events in individual cells in real time. Nanotechnology provides additional tools to visualise single molecules.

The aim of our project is to build on our existing imaging capabilities in the Centre for Cell Imaging and our world-leading expertise in the synthesis and imaging of nanoparticles to improve the equipment available with the most up-to-date microscopes. We will also develop new technologies, software and image analysis methods that will allow us to increase the resolution of the images and their interpretation (to better see the detailed structures inside the cells). We will make sure that the equipment is used to the best of its capabilities to answer important biological and biomedical questions. For this purpose, we will select the best projects, in Liverpool, the UK and beyond, and we will support users from the conception of their experiments to the analysis and interpretation of their results.

The CCI platform will therefore permit researchers from a broad range of research fields to tackle important 21st century challenges in biomedical research, by investigating in real-time cellular events at the molecular level. To illustrate the breadth of science that will be served by the platform, we briefly present below three exemplar projects that, among many others, will benefit of the improved facility.

UNDERSTANDING ACUTE PANCREATITIS ON THE NANOMETRE SCALE. Cells of the pancreas produce the enzymes that digest our food inside the intestine. In acute pancreatitis, instead of being secreted to the intestine, enzymes become active in the pancreas cells themselves and damage the pancreas leading to severe pain, hospitalisation and in some cases, death. The activation of these enzymes involves the formation of dynamic structures that are very difficult to study by conventional microscopy because they are smaller than the resolution of conventional microscopes. The new platform will uniquely allow us to study the response of those structures when pancreatic cells are exposed to molecules that may induce or prevent acute pancreatitis and ultimately develop novel approaches to prevent this from happening.

IMAGING THE SUBTLE ROLE OF OXIDATIVE STRESS IN AGEING; Oxidative stress leading to oxidative damage has been implicated in the processes underlying ageing for over 50 years, but recent data demonstrated that rather than gross changes in oxidative stress damage, it is the more subtle changes in regulation of reduction-oxidation reactions that play a fundamental role in ageing processes. The new platform will uniquely allow us to control and measure oxygen level and oxidative stress providing new insights into mechanisms of ageing.

PROBING THE MECHANISM FOR INTESTINAL ABSORPTION OF NANOMEDICINES; Insolubility of active pharmaceutical ingredients and the resulting lack of bioavailability is a major concern for the development of new drugs. Approaches based on nanotechnology have proven successful, but very little is known regarding the mechanistic basis. The present application will enable us to study differences in trans-intestinal passage and toxicity of nanomedicines compared to conventional dissolved drugs.

The aim of Liverpool Imaging Partnership is to support excellent science with state-of-the-art live cell imaging. We will build on our expertise in long term live cell imaging and single molecule imaging using nanoparticles, to measure intracellular molecule dynamics in a wide range of living models including cells, tissues, small embryos and plants. To improve the sensitivity of detection and accurately measure protein-protein interactions, we will install a LSM 780 (Carl Zeiss) with Fluorescence Life time imaging (FLIM) component. The considerable increase in sensitivity of the LSM 780 allows it to be used in photon counting mode (FCS/FCCS). This will provide capabilities to measure intracellular sensors (e.g. oxygen nanosensors), protein-protein interactions, protein diffusion and complex formation in living samples. It will have direct application in several projects, such as elucidation of mechanisms of pancreatic damage, testing drug specificity. To increase our resolution capabilities and correlate intracellular dynamics with ultrastructural changes, we will develop two new techniques: 1) super-resolution optical fluctuation microscopy (SOFI), which requires a limited investment in comparison with other super resolution techniques and logically builds on our expertise in quantum dot development; 2) photothermal-correlative light microscopy (p-CLEM) where the gold standard of EM probes becomes also an excellent optical probe. This will constitute a platform with high sensitivity state-of-the-art confocal microscopes coupled with photothermal imaging of nanoparticles that is dedicated for long term imaging and imaging in physiologic settings including oxygen controlled environment. Altogether, this will provide unique measurements in living cells supporting research in areas such as inflammation, ageing, cancer, drug safety, nanomedicine, plant biology, stem cell research etc. in a platform that will support users from experimental design to image analysis.

1. The Partnership will first impact on the scientific community by providing new tools for cell imaging. This will primarily impact on the research developed at the University of Liverpool, but also on other UK and overseas Universities (see letters of support). The strengthening of the CCI will provide for a step change in the ability to measure intracellular events at the molecular level, which will synergise with the existing research base. This will ultimately have impact on society by providing new knowledge, some of which will be critical to translational projects. Important beneficiaries will, therefore, be academics and clinicians. Examples include the MRC-funded Centres for Drug Safety and Integrated Research into Musculoskeletal Ageing and NHS stakeholders, such as the NIHR-funded Pancreas Biomedical Research Unit, Phase I/IV capability and future (MHRA accreditation due October 2012) Phase I trials unit.
2. The Partnership will also have economic impact, since IP will be actively protected and routes to exploitation pursued. For example, DF has patented novel stabilisation and functionalisation of quantum dots and nanoparticles for use in sensors and diagnostics; SR is co-founder of two spin out companies, IOTA NanoSolutions Limited and Hydra Polymers Limited. Our contribution to the development of SOFI, which is a versatile approach to super-resolution, will support its adoption and implementation by a large body of users and, will have direct (low cost super-resolution, development of new software in collaboration with Carl Zeiss through a case studentship) and indirect (via the discoveries enabled by SOFI) economic impact. The Liverpool Imaging Partnership will facilitate the training of the next generation of scientists in a multidisciplinary and vibrant environment to ensure the excellence of the UK science base.
Way to Impact: To achieve this economic impact, the University has a dedicated team, Business Gateway (BG), which aims to facilitate links between the university and companies and other external organisations. At its inception the Partnership includes new collaborations with RedX pharma and with Promega, as well as a substantial deepening of the collaboration with Carl Zeiss, and, with the support of the BG, we will organise annual outreach activities combined with our imaging day, aimed at broadening the industry user base.
3. The partnership will contribute to tackle important biomedical challenges, which will impact on society. Improvement of drug safety and use of nanomedicine, as well as increase understanding in pancreas disease, malaria, cancer progression and ageing will ultimately improve drug development for major disease treatment and hence well-being and health in the population. To maximise the impact on society and public awareness, the investigators will be involved in diverse communication activities, which were recently recognised by a BBSRC Excellence with Impact prize to the Institute of Integrative Biology in 2011.
Way to Impact: We have experience of staff visiting local schools, providing exposure to modern biology and encouragement for science careers (participation to Science fair by RL). Members of staff also attend University Open days, where they encourage young students to science. The CCI platform is one of the facilities shown to pupils and their families and provides very accessible visual tools for explaining scientific development. A further stage is through work with local schools and colleges (e.g. St Edwards College; Winstanley College; Range High School; Shorefields Technology College, Priestley College Warrington, the Princes Trust/ Mersey Fire and Rescue) and to give summer studentship placements supported by Nuffield Bursaries (RL, VS, AH, DB). Wider outreach ranges from a special "Murder Mystery" event at the Liverpool World Museum UoL (AH). RL has given several Christmas lectures for six-form students. These activities will be continued and expanded.