Super-resolution imaging

Bioimaging, the investigation of cells and organisms by microscopy, is one of the key technologies for life science research. Often the way life functions has however been studied in fixed samples and by reducing cells and organisms to 2-dimensional projections. Although this approach has led and will also in the future lead to breakthrough discoveries there is a realisation that if possible "life" should be studied in a live 3D environment. Access to live cell imaging, the investigation of live cells or organisms by microscopy over time, thus is critical for state-of-the-art life science research. Live cell imaging becomes the more powerful the smaller the structures are that it can distinguish or 'resolve'. Diffraction of visible light generally limits the ability of imaging system to resolve structures to a size of 1/50th to 1/100th of a cell diameter. For a further gain in resolution a number of 'super-resolution' microscopy approaches have been developed over the last few years. Many of these approaches are however too slow to capture the rapid dynamics of life cells and organisms. A fast super-resolution approach has recently been made commercially available, called structured illumination microscopy (SIM) with lattice illumination, that is much better suited for the imaging of live cells.
We apply for a state-of-the-art live cell imaging instrument with lattice SIM, the Zeiss Elyra7. Its live cell super-resolution capability is complemented by the ability to detect even smaller structures, single molecules, in fixed cells. We will integrate the technology into the Wolfson Bioimaging Facility, the centralised microscopy facility of the Faculty of Life Sciences at the University of Bristol. This facility provides access to and technical support for a large number of imaging technologies for 100s of members of staff at the University of Bristol and dozens of outside collaborators. With the Elyra7 we will, for the first time, be able to offer rapid super-resolution live cell and single molecule microscopy. To the best of our knowledge, these would be unique capabilities in the entire Southwest of the UK. The Elyra7 will be used immediately by a core group of 13 heavy users at the University of Bristol that support this application. We will implement a detailed strategy to grow use at the University, in the greater Southwest and in our user base in industry. The Elyra7 will thus enable and enhance a large number of research projects.
We will apply the Elyra7 to study many research questions in live cells. These include subcellular trafficking, the movement of molecules and subcellular structures such as vesicles within a cell, and the regulation of the behaviour of various cell types that need to rapidly adapt to changing physiological conditions within our body, such as neurons and blood cells. We will also use the microscope to advance our understanding in Synthetic Biology. Some of our Synthetic Biology research focuses on the design of new protein molecules that can be used to regulate cell behaviour, for example as a platform for vaccines. Our basic research is closely linked to translational exploitation, through industrial research collaborations and a number of University spin out companies. Super-resolution microscopy will strengthen research in these collaborations and therefore generate a larger economic impact.
Training of the next generation of scientists is an important aspect within the Wolfson Bioimaging Facility as it supports a large number of Ph.D. training programmes. Training in the cutting-edge live cell imaging approaches enabled by the Elyra7 will be integrated into our Ph.D. programmes. Many undergraduate students, from the University and beyond, also participate in microscopy-based research enhanced by the Elyra7. Striking images enabled by the super-resolution capabilities of the Elyra7 will strengthen outreach to secondary school students and the wider public.

Live cell imaging is critical for capturing dynamic biological processes. It is particularly powerful at high resolution (120nm), at high speed (ms acquisition times) and in two or more colours to investigate colocalization. Here we apply for an advanced super-resolution imaging system for the Wolfson BioImaging Facility (WBF) at the University of Bristol. The WBF supports >390 investigators, internal across 4 faculties and external. The lack of live cell super-resolution imaging severely limits many research projects relying on the WBF. The requested system provides super-resolution imaging using structured illumination microscopy as recently advanced through pioneering lattice illumination and rolling interference-based image generation. The system can achieve super-resolution to 120nm with acquisition speeds to 5ms. Complementing live cell imaging, the system supports single molecule localisation microscopy with an even higher resolution of 20nm, mostly in fixed samples. With great flexibility in adjusting to the competing demands of resolution, speed, depth and field of view the system is well suited for super-resolution imaging from molecules to organisms as covered by the wide user base of the WBF. The system will thus enable cutting-edge super-resolution imaging for potentially hundreds of researchers across the University of Bristol and beyond. There already is a group of 13 "core" users for the instrument with research spanning cell biology, neuroscience, immunity and synthetic biology, as described in the research plan. A key impact objective of this application is to grow our already extensive external userbase with emphases on the GW4 University alliance (Exeter, Bath, Cardiff) and users in industry. The imaging system will also support training at the undergraduate and postgraduate level and as a leading technology piece of equipment provide a strong career development opportunity for our technical staff.

This application brings super-resolution live cell imaging and single molecule localisation microscopy to an extensive user base, >390 current users of the Wolfson BioImaging Facility (WBF), including external academics and users in industry.

With a group 13 initial heavy users in place, our first impact objective is to expand this user base within the University of Bristol (UoB) and in the region. With user fora, newsletters and a dedicated inter-university workshop we will engage potential new users at the UoB and our GW4 university alliance partners in Bath, Cardiff and Exeter. Our GW4 partners can access the WBF on UoB terms. We will also continue close engagement with Bioimaging UK and the Royal Microscopy Society to ensure potential UK users are fully aware of our capability and their opportunities for interaction here. Publicity through webpages, mailing lists and/or existing Wiki will continue.

A substantial fraction of WBF users, including many of the applicants here, collaborates with industry through research spin out companies, CASE studentships and research contracts. Many of these collaborations, e.g. work by Woolfson on vaccine design and Wuelfing in immuno-oncology, are driven by the scientific excellence of UoB staff in investigating dynamic cellular behaviour with an array of leading imaging approaches not available at our industrial collaborators. Super-resolution imaging will thus enhance industrial research contracts and work across eight recent spin out companies from the UoB Faculties of Science and Life Science. In addition, eight industrial partners not directly related to research at the UoB, mostly regional SMEs but also a leading pharmaceutical company, are users of the WBF. We will contact the industrial partners that are most likely to benefit from a super-resolution system. We aim to further expand our industrial user base by involving our Knowledge Exchange Officers in Research and Enterprise Development and Innovation Fellows in Schools and Faculties and by including super-resolution imaging into on-going industrial outreach events.

Our work addresses the basic function of eukaryotic cells in research areas such as cancer biology, immune signalling, neuroscience, osteoarthritis, plant defence to pathogens, platelets, synthetic vaccine platforms and viral infection. These research areas link basic research to future translation upon involvement of industrial partners. While outside of BBSRC remit, this substantial translational potential must be considered in the context of long-term societal impact.

Skill development is a key impact objective. By introducing super-resolution imaging to dozens of undergraduate project, M.Sc. and Ph.D. students across our laboratories and by including it into introductory session of eight Ph.D. programmes at the UoB we will teach a new generation of scientists and develop their careers through acquisition of cutting-edge skills. Our projects are also data intensive, and the management and analysis of such large datasets is applicable to many areas of professional life.

Public engagement is a key impact objective with a focus on secondary school students and the wider public. The applicants, the WBF and the wider UoB continuously engage in a wide range of outreach activities, as detailed in the Pathways to Impact, into which we will rapidly integrate a super-resolution imaging system. Super-resolution live cell imaging data has outstanding visual appeal and we will prominently feature it across our wide range of outreach activities to entice secondary school students to pursue a career in science. Similarly, the visual impact of the work described in this application provides an optimal way to engage the wider public. As an example of a prominent outreach event, within two years of the installation of a super-resolution imaging system the WBF will organise a public lecture on 'seeing cells in action to discover how our bodies work'.