Photo-oxidation and cryofluorescence for Correlative Light Electron Microscopy.

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Considerable technical developments have enabled us to image live cells to gain incredible detail of time-sensitive cellular events (on millisecond time scales). Advances in electron microscopy mean that we can image events at very high resolution, but only in fixed, processed samples. This can achieve 3D reconstructions of cellular structures at nanometre resolution. Combining these approaches, a technique called correlative light electron microscopy (CLEM), allows us to achieve high resolution movies of cell dynamics and high resolution ultrastructure of the same events. The University of Bristol is at the forefront of developing methods for CLEM, making us ideally placed to exploit new technologies in this area. New developments allow combined live cell fluorescence imaging and high resolution ultrastructure imaging by electron microscopy using a single probe. Excitingly, these same probes can also be used for proteomics experiments. Proteomics provides methodologies to catalogue and quantify all proteins within a sample. These new probes allow us to move towards our long term goal of combining imaging with proteomics. We would now be able to express a probe to label a specific sub-cellular compartment. We would then be able to define its dynamics by live cell imaging, its ultrastructure in 3D using electron microscopy, and to label all proteins within the spatially restricted area of the probe prior to proteomic identification. This has enormous potential to define how the composition of protein machines changes as structures move, change shape, or mature into different forms. These new approaches require specialist equipment for both modes: high sensitivity and specifically configured light microscopy equipment, combined with specialist sample preparation and imaging equipment to prepare samples for electron microscopy. We are in the fortunate position in Bristol of having outstanding facilities for imaging and proteomics that are used heavily by researchers in Bristol as well as across the UK. Thus, the costs of providing additional equipment to link LM, EM and proteomics are not as high as they would be if starting from a less well equipped centre. We propose projects within this application that range from fundamental studies into complex cell biology through to experiments to examine interactions of cancer cells with the immune system, the development of the skeleton, and the way in which newly designed nanostructures interact with cells. Our work has implications for multiple areas within BBSRC remit including basic bioscience underpinning normal human and animal health, infection, and aging. The portfolio of projects includes researchers with a strong track record of BBSRC funding and covers areas of direct relevance to BBSRC remit and strategy, as well as early career researchers whose work is developing in similar directions. The cohort of applicants provides a showcase for the future possibilities of this work from which we expect to derive significant additional use from within Bristol and beyond. Indeed, the early phases of this work have already attracted significant external interest through major international Bioimaging schemes. This work will also be a partnership with Leica Microsystems who have a strong track record and ongoing interest in commercialization of these methodologies; this ensures future technical and commercial development. While embedded in existing technology, this proposal therefore has significant impact potential to the industrial sector as well as from the bioscience research itself.