Photo-oxidation and cryofluorescence for Correlative Light Electron Microscopy.

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.

Our application seeks to implement newly developed technologies for correlative light electron microscopy. These experiments will use a state-of the art scanning confocal microscope equipped with high sensitivity detectors. Live cell imaging will capture events on fast timescales and at the opportune moment we will trigger conversion of the fluorescent signal to an electron dense precipitate. This will be achieved using a fluorescent singlet oxygen generator (miniSOG) where activity is triggered by photo-oxidation microscopy using a specially configured photo-oxidation microscope, or using an enzymatic method based on engineered ascorbate peroxidase (APEX).
Tagged proteins will be used in cells and zebrafish embryos for live cell imaging. At the very point where we observe an event of interest e.g. a specific cell:cell interaction, maturation of an organelle, or a change in morphology, we will use photo-oxidation or chemical peroxidation to transform the probe into a singlet oxygen generator to precipitate diaminobenzidine. Photo-oxidation will be achieved on a dedicated system with controllable laser illumination and a cooled stage for control of the precipitation reaction. Accurate correlation of fluorescence and EM images requires that we can image frozen sections at cryo-temperatures. The requested cryofluorescence system is newly developed by Leica and is available to us prior to release.

The technology that we propose to implement has the added advantage of enabling us to take steps toward spatially resolved proteomics. Organelle markers will be tagged with APEX, used for CLEM using DAB as a substrate, and in parallel for selective biotinylation of proteins in close proximity using biotin-phenol as a substrate. Biotinylated proteins will be detected using quantitative mass spectrometry. The combination of CLEM and proteomics here has the exciting possibility for us to define protein machines involved in spatially and temporally restricted events.

There are key aspects within the project that have potential to be of use in the development of technologies within Bioimaging and related industries. There is great interest in the possibilities of CLEM and our collaboration with Leica in this project will ensure rapid dissemination and even possible commercialisation of core technologies. While not a technology development proposal per se, as with any cutting edge technique, there are future commercial possibilities. One immediate possibility here could be through the adaptation of existing systems to accommodate laser-based photo-oxidation.

With the individual research projects there is significant potential for impact in the longer term. Our work addresses the basic function of all mammalian cells which therefore underpins our understanding of the healthy organism and age-related changes. Other work within the proposal seeks to gain a better understanding of the cell biology of the important zoonotic pathogen Salmonella. Furthermore, a higher temporal and spatial understanding of cancer cell biology, synaptic function, immune cell contacts, and skeletal development and dysfunction highlights the importance of a full understanding of these pathways to guide possible future clinical intervention. While outside of BBSRC remit, these more clinical possibilities must be considered in the context of long term impact. While it is always more complex to define the way in which and timescales for such impacts might occur, we can develop such lines through our impact plan. Through informing our basic understanding of a critical cellular process, it is most likely our work will inform long term projects in other fields including the pharmaceutical industry.

Potential applications of this work are identified from within the labs involved as well as by continuing liaison with our Research and Enterprise Department. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders such as BBSRC when appropriate. As with all of our projects, this one includes considerable opportunity to train the researcher involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes, the extensive network of University schemes to benefit the training and development of research staff (Bristol is at the forefront of research staff development).

Several applicants have good track records in facilitating the placement of staff in areas outside our core research activity. For example, a previous postdoc in the Stephens lab undertook a period of flexible working in order to shadow some of our Research and Enterprise team and subsequently undertook a part-time course in intellectual property management and now works full-time for a company in this role; similarly Martin has facilitated the movement of a graduate student to a career in science communication (now performing this role at Columbia University in New York). This demonstrates that the environment as a whole is highly conducive to career development of our staff beyond academic, basic science research alone and thus contributes to the economic development of the nation. Our projects are also very data intensive- notably from imaging work - and the management and analysis of such large (terabyte) datasets is applicable to many areas of professional life. This work will lead to significant image data that is readily used in both public understanding of a science and artistic arenas. Examples include local exhibitions and promotions. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore is likely to contribute to local exhibitions or displays as has been the case with previous work from our labs and others within UoB.