MICA: Nanoscopy Oxford (NanO): Novel Super-Resolution Imaging Applied to Biomedical Sciences

Over the past decade entirely new technologies have been developed that bypass the limits of light microscopy allowing researchers to visualise, for the first time, intracellular structures, the cell surface and the extracellular space at the nanometre scale in both fixed and living cells. This has created entirely new possibilities to investigate normal cell structure and function. The principal aim of this application is to fully integrate and coordinate multidisciplinary groups (biologists, physicists, engineers and chemists) across the Oxford campus, to develop super-resolution microscopy (SRM) and to make this available to a broad biomedical community (e.g. WIMM, Dunn School, WTCHG, Biochemistry and DPAG) to generate new insights into normal biological processes and how these are perturbed in human diseases. We propose that this new consortium is called Nanoscopy Oxford (NanO).

Over the past few years several biomedical institutes and Departments in Oxford have made commitments to developing imaging as a key infrastructure for supporting their scientific programmes. In addition these departments and institutes have contributed to an overarching strategic vision for imaging in Oxford under the leadership of the Micron initiative. This has enabled the Oxford campus to establish a wide range of conventional imaging and to take the first steps in developing SRM. Recruitment of scientific leaders in this area (e.g. Davis, Eggeling, Schermelleh) and their collaborations with international leaders in this area of research (Hell, Sedat, Jakobs) have placed our consortium in strong position, with further investment, to become a leading UK and international centre for next generation imaging.

Recognising that no single approach is suitable for all applications, we will build upon current imaging facilities and associated management structures in Oxford to establish, extend and optimise the full range of approaches to SRM (STED, RESOLFT, 3DSIM and PALM/STORM). Furthermore, we will adapt each type of microscopy to maximise our ability to perform nanoscale imaging, developing new approaches for multi-colour and three-dimensional imaging. We will improve the quality of imaging in thick specimens by reducing aberrations, improving fluorescent labels for live-cell SRM imaging, and we will speed up the imaging process and develop new methods for analysis of the super-resolved images. By streamlining these techniques we will bring complex technologies, not yet commercialised, within the reach of biomedical researchers with limited experience of SRM.

The consortium has well established programmes in teaching and training biomedical researchers in the use of all forms of microscopy and therefore is well placed to become a training facility for the UK in this area. Realising this vision including technical development, adoption of techniques to analyse key cell biological questions and then applying them to biomedical issues (fully integrating Micron and NanO) will make Oxford an international centre of excellence in the development of next generation microscopy and its application to the analysis of human disease.

The past decade has seen a major revolution in optical microscopy, with new methods surmounting the conventional diffraction limit in spatial resolution. Super-resolution microscopy (SRM) techniques such as stimulated emission depletion (STED), REversible Saturable/switchable Optical Linear (Fluorescence) Transition (RESOLFT), (Saturated) Structured Illumination Microscopy ((S)SIM) and (fluorescence) PhotoActivation and Localization and (direct) Stochastic Optical Reconstruction microscopy (PALM/(D)STORM) permit imaging of living cells with increasing resolution and are opening a broad frontier in the study of cellular functionality. Each of these methods has distinct advantages and disadvantages for imaging different types of specimen. The principle aim of this proposal is to make all developments in SRM available to the biomedical community in Oxford to enable unique insights in cellular research. We already have a well-established infrastructure to support advanced optical imaging within the University with the Micron Advanced Bioimaging Unit and planned facilities in the WIMM and at WTCHG. There are two SIM platforms in Micron but we will establish STED and RESOLFT imaging at the WIMM and provide access to a home-built PALM/STORM system in Micron.

SRM techniques have enormous potential, yet their utility is still restricted by a number of inherent limitations. A second aim is to grasp this opportunity to unite biologists with physicists, engineers and chemists to drive further developments in the technology of SRM imaging. We will develop:
1. STED and RESOLFT approaches in Oxford.
2. SSIM with fast image acquisition at a spatial resolution of 50nm for 3D imaging.
3. adaptive optics for the correction of aberrations encountered when performing SRM at depth within tissue.
4. live PALM/STORM and FRET.
5. new image analysis and control algorithms for the super-resolved SRM data.
6. fluorescent labels for live-cell SRM.

Nanoscopy Oxford (NanO) will be a hub for advanced and innovative imaging nationally and internationally, by fostering not only an interactive and collaborative environment within Oxford University, but also developing much closer links between different research groups in academia and Industry in the UK and abroad. This strategic vision for the future will continue to keep Oxford at the international forefront of this increasingly exciting phase of biomedical basic and translational research.

As explained in the "academic beneficiaries' section, we can be certain that the technological developments and facilities that we plan to establish throughout the next five years will be available to and benefit biomedical researchers worldwide. The following groupings will benefit from the technology development and from specific discoveries that will be made by applying the new technology to leading edge biology.

1) All researchers working at the interdisciplinary interface between physics and biology, developing next generation optical microscopes and methods of super-resolution microscopy (SRM). The progress we envisage will provide insights into future developments and applications.
2) All biological researchers applying advanced imaging methods at the limit of what is currently possible by giving them access to new technology and/or by directing their research with published insights gained within NanO.
3) Chemists and companies working on new fluorescent labels such as organic dyes and fluorescent proteins and labelling protocols, which are optimized for SRM and optical microscopy.
4) Companies developing optical microscopy by improving their basic technology and allowing them to stay highly competitive. For example: Leica Microsystems, Carl Zeiss Microscopy, Nikon Microscopy, Olympus Microscopy, Applied Precision/GE Healthcare, Perkin Elmer, PicoQuant and Abberior Instruments.
5) Companies performing biomedical research and high-throughput-screening based on optical fluorescence microscopy by offering them novel, improved technology platforms. For example: Pharmaceutical and biotechnology companies such as GlaxoSmithKline, Pfizer, Novartis, Lilly, Evotec. Our initiative will contribute to ensuring a high educational level for the future workforce of this important sector of the economy.
6) UK research in general by establishing an internationally competitive platform for next generation optical microscopy and attracting the brightest minds from all parts of the globe.
7) Contribution to public health and public welfare by gaining further insight into the mechanisms underlying normal cell biology and human disease. We anticipate that application of SRM imaging research in crucial areas of experimental medicine (such as immunology, haematology and neuroscience) will lead to greater insights into the mechanisms causing many human diseases hence providing opportunities to identify diagnostics and therapeutics.
8) Enhancing the knowledge-based economy by improving the training of highly skilled researchers in the development and applications of the new technology.
9) Attracting new industrial R&D investment in the UK by enhancing cutting-edge research at UK universities and research institutes.