Correlative In Vivo Fluorescence and Micro-Computed Tomographic Imaging of Tissue Structure and Function

For human and animal bodies to function properly, the tissues and organs that make them up must be organised properly (have the correct tissue architecture). This must happen across many size scales, from individual cells to large organs such as the liver, kidneys, and bones, or blood vessels and nerves supplying the organs. Given cells and tissues are dynamic three-dimensional (3D) structures, we need to see how they fit together in 3D to understand their architecture, how structures connect and vary within the tissue. For example, in Regenerative Medicine, we try to make tissues and organs to repair damaged and diseased tissues and restore the body to its original health. Scaffolds upon which the cells can grow and are guided are used to help organise the cells into the right structures. We are now in a unique position to create new soft and hard tissues (e.g. liver, neural tissue, cartilage, bone) to improve treatments and to better understand normal and abnormal human and animal body function. Similarly, in aging the composition of our bodies changes, and it is important to be able to track and understand how this happens in living animals, so that ultimately we can have the knowledge to better treat a range of diseases, like heart disease, lung disease and cancer.

We are applying for an imaging system that will allow us to examine cells, organs and tissues within the body (called Correlative in vivo imaging) to create 3D images at successive time points, without having to kill the animal to look inside it. The system will allow us to label and follow specific cells and molecules within the body and to create 3D image stacks of tissues of interest with a high-resolution micro-computed tomographic (uCT) imaging device. This uses X-rays to non-destructively image large samples and the design of this new instrument allows us to distinguish individual tissue components and how some of their features change over time, in a way that is not currently possible in two dimensions (2D) through a technique called histology, using normal, light microscopy.

The imaging system will add to the wide range of existing imaging facilities in Southampton that are supported by 12 expert imaging staff, employed at the Biomedical Imaging Unit (BIU) and the u-VIS X-ray Imaging Centre (u-VIS). The existing expertise in sample preparation and biological image interpretation is essential for these cutting edge imaging techniques to be used effectively. An additional problem in 3D imaging at all scales is the very large digital image datasets that are produced, each taking large amounts of storage, typically; 50-1000Gb (equivalent of 10-200 DVDs each!). On this account, it is pivotal to have dedicated staff in place who will run and make sure that the imaging system performs efficiently and can provide a reliable, streamlined service to all users.

The UoS has agreed to fund a Research Support staff member to underpin and provide a sustainable Imaging Service. Provision of the combined preclinical correlative in vivo imaging system, together with our existing leadership team in advanced computing and image processing (state-of-the-art computing hardware, software and expertise already in place at BIU and u-VIS, will allow investigating and understanding tissue architecture and to understand better tissue function.

In this project, we will work with our collaborators in the University to develop further correlative in vivo imaging application areas of tissue samples, specifically including processing and analysis workflows for the 3D datasets. Our ability to understand tissue development and formation in its native 3D context has the potential to transform human health over the next 10-30 years. In order to fulfil this promise, as quickly and safely as possible, it is essential we can image the generated structures and tissues in vivo in a longitudinal fashion and at different hierarchical levels of tissue organisation.

Medical advances in the developed world have been linked to sustained increases in life expectancy. This progress presents its own new challenges: increases in age-related pathobiology and associated reductions in quality of life have substantial socio-economic costs. Complementary, multi-scale and multimodal in vivo assessment methods are required to interrogate cell fate, development and cell/tissue function and dysregulation, for example in ageing. As living tissue is inherently 3D and dynamic, a comprehensive quantification of cell interactions, cell differentiation and tissue architecture and function requires analytical 3D imaging methods. These require appropriate spatial resolution, structural differentiation, volumetric analysis capabilities and a capability for providing co-registered and spatially-resolved readouts for tissue function. Time-resolved, in vivo 3D imaging of specific fluorescently labelled probes provides an essential further dimension (i.e. '4D' imaging) not available through structural investigations alone. A key unmet challenge at the UoS is to track tissue architecture non-destructively in living animals, allowing higher fidelity, contextually relevant information to be gathered from fewer experimental animals with applications throughout the biological sciences.

Southampton Correlative In vivo Imaging comprises an integrated, non-invasive, non-destructive Imaging system to track the spatial and temporal distribution of tissue structures and physiologically relevant labels in tissue-engineered constructs, tissue samples in live animals

The microCTHR/FLR system from MILabs (preferred platform), a multispecies preclinical imaging system, offers longitudinal in vivo imaging capabilities, in addition to ex vivo sample scanning. It is important to note that this system allows not only imaging of hard tissues such as bone, but also soft tissues, including lung, liver, adipose tissue, vascular systems or cancerous tissue.

Health and Science: The aims of the project, and the impacts arising from it, directly address the BBSRCs strategic aims as outlined in Forward Look for UK Bioscience, with particular focus spanning the three main themes (e.g. Transformative technologies (Theme 1); Bioscience for an integrated understanding of health (Theme 2) and Building strong foundations (Theme 3). Our proposal aims to provide a central resource for longitudinal, non-invasive imaging of health in living animals, bridging diverse research fields in medicine with those in the physical sciences

Our Correlative In Vivo Imaging platform for UoS and wider UK Research will substantially impact on the ability to provide and apply best cutting-edge practice within in vivo 3D imaging. Project outputs will have significant bearing on the quality of life through enhanced knowledge of biological and physiological and improved regenerative medicine capability and for researchers engaged in biological science across strategically important areas such as developmental biology, cardiovascular sciences, neurosciences, cancer sciences, respiratory research and biomaterials and implant sciences. The same specialties, outside UoS will benefit. Concurrent with this, we aim to promote and foster the development of technical staff - their transferable skills and employability beyond academia - as well as maximising research impact beyond the basic science. This will include drug and instrument development, impact in public policy though underscoring importance of preclinical animal models in research, as well as public understanding of a wide and varied science base at the university.

National Network: The microCTHR /FLR combined CT and optical imaging in vivo capability integrated with our world leading Biomedical Imaging research facility enabling multidisciplinary open-access imaging service for biological, developmental, regenerative and bioengineering science imaging, would provide a step change for the UK Research Community. Our proposed in vivo capability, building on Southampton Imaging (UKRMP I) will facilitate collaboration within UKRMP II and with non-Hub groups (i.e. Regenerative Medicine Community) as well as FortisNet, a collaborative network in musculoskeletal (MSK) health launched in 2016 (Smith inaugural Director and Oreffo co-I). The FortisNet partnership has established regional and national collaborative relationships in MSK health between industry, the NHS, stakeholders and academic researchers. Application of real time correlative in vivo imaging and optical imaging will be made available to our FortisNet Group enhancing and developing additional collaborations and research platforms.
Animal Use Reduction: Real time and correlative in vivo analysis, using the same animal, will facilitate a dramatic reduction in animal use for in vivo studies meeting and addressing 3Rs objectives for UK science. Our strategy is to share our data and the approaches developed and used for analysis with the wider scientific community to provide immediate access, enhancing collaboration and research.

Research Knowledge & Training: We will disseminate results at various science forums. We have collectively presented keynote and plenary lectures at over 27 meetings around the world in the last 24 months. As part of our established Excellence with Impact programme, we will build on links with Business Fellows to promote innovative collaborations with industrial partners, FortisNet (see letter) and SMEs. This equipment grant will contribute substantially to the training and development of the research community. In order to maximize broader impact to the general public, we will organize a number of educational outreach programmes (We already have funds as part of UKRMP II Acellular SMART materials Hub for knowledge dissemination) building on our links to a new school education programme, the LifeLab Southampton (https://www.southampton.ac.uk/lifelab/index.page).