Cryo-fluorescence Tomography - a new paradigm for ex vivo imaging

Aims and Objectives Aim: We seek to establish an EMIT Imaging “Xerra” cryo-fluorescence tomography (CFT) platform within an open access, core imaging facility at the University of Southampton (UoS) which specialises in multimodal, multidimensional, multiscale and correlative imaging and supports its users from project design to publication and dissemination. Objective 1: To satisfy unmet need to enable fluorescent markers to be localised in 3D within ex-vivo animal tissue with high resolution and sensitivity. Objective 2: To bridge the significant gap in resolution, sensitivity and information potential that exists between our in vivo optical imaging systems and our high-end fluorescent microscopy systems. Objective 3: To make immediate and substantial impacts for multiple research teams from across the South Coast engaged in BBSRC’s strategic objectives, including its Advancing Frontiers of Bioscience and Tackling Strategic Challenges portfolios. Context CFT is a revolutionary new technology that combines the benefits of serial block face imaging with fluorescence imaging. It automatically carves thin slices away from the surface of deep-frozen biological samples (tissue, organ or whole animal – up to the rat size) and images each newly-exposed block face under multiplex fluorescence (with multiple exposure times for high dynamic range) and white light to detect fluorescently-labelling within its anatomical context with nM sensitivity and high resolution (isotropic 20-55 µm). Tomographic reconstruction converts slice data into 3D data, illuminating probe distribution throughout the entire sample with minimal sample preparation. The process can be paused to allow tape film to be applied to the block face, allowing the next slice to be recovered for downstream analysis. Currently CFT is only commercialised by one manufacturer (the EMIT Imaging Xerra), with a handful of systems installed globally in world-leading institutions. Introducing this technology to UK academia through an open access core facility has huge benefits. Research enabled CFT has immediate and wide application across Southampton’s bioscience portfolio for visualising and analysing fundamental biological process in health and disease including tumour growth and metastasis, angiogenesis, immune cell distribution and migration, brain drainage pathways, expression of fluorescent protein markers, analysis and optimisation of targeted therapeutics and drug delivery systems and detection of off-target accumulation and effects. Essentially anything requiring ex-vivo analysis of endogenous or applied in vivo fluorescence within small animals will benefit from 3D analysis with greatly-enhanced resolution and sensitivity. Application and Benefits (1) Fostering collaboration and sharing of new technology: with only one (commercially-owned) Xerra installed in Europe (London), establishing CFT in an open-access university imaging core, has local, regional and national impact. Researcher co-Leads are drawn from four South Coast institutions to evidence the need. (2) Equipping the next generation: a core facility installation allows the widest range of undergraduate and postgraduate students, postdocs, early career researchers (ECRs), academics and Research Technical Professionals (RTPs) to benefit through mentored access, training, and ongoing advice and support from project design through to publication. This promotes transferable skills and CPD and enhances UoS’s many doctoral training programs, equipping research teams with expertise to drive innovative solutions and advances in biological and biomedical science. (3) 3Rs: although CFT samples come from animal experimentation, it maximises the information gathered from each animal in furtherance of the 3Rs. Data of a higher standard requires fewer animals to be statistically significant, thus CFT has the potential to reduce the numbers of animals used.