Super Resolution Imaging for Cell Biology and Neuroscience at UCL

Microscopy has been one of the principle tools for discovery-based research at the cell and molecular level, allowing insights to the organisation and dynamics of cells, as well as the faults associated with infection and disease. Over the last 25 years increasingly sophisticated microscopes, together with new experimental tools such as fluorescent protein tags, improved lasers and cameras, and developments in live cell imaging, have led to rapid advances in our understanding of cell organisation and function. Nevertheless, imaging at high resolution is limited by the properties of light to ~250 nm, meaning that information at the level of molecules and molecular complexes cannot be obtained using conventional light microscopes. Thus, high-resolution studies have been restricted to electron microscopy (EM), which can only be used on fixed and highly processed samples, and has limited capacity to identify specific molecular components.

In recent years several novel optical and computational approaches have been developed to break the diffraction-limit of conventional light microscopy, with the potential to obtain molecular/structural information at significantly higher resolution (approx. 2-20 fold improvement). By combining multiple fluorescently-labelled probes, within the same sample, together with live cell imaging, these so-called super-resolution nanoscopy techniques are able to bridge the current technologies of diffraction-limited microscopy and EM to generate new detailed molecular and mechanistic insights into the cellular and molecular processes that underlie normal cell function and disease.

Super-resolution imaging (SRI) instruments are now becoming commercially available allowing cellular biomedical researchers to begin to apply these exciting new technologies to a multitude of questions in cell biology, neurobiology, infectious disease and many other fields of biomedical research. Development is still at an early stage and there remain many opportunities for progress, particularly in live cell imaging, probe development, software development, analytical tools, etc. However, SRI instruments and technical developers are needed now to work in close collaboration with biomedical researchers to establish the technology and push instrument and application development.

This application will link MRC-funded scientists in the Cell Biology Unit/Laboratory for Molecular Cell Biology (CBU/LMCB) and Prion Unit (MRC PU), as well as the MRC/UCL Centre in Molecular Medical Virology (MRC CMMV), with members of UCL's Faculties of Life Sciences, Medicine and Brain Sciences, the London Centre for Nanotechnology (LCN), UCL physicists and chemists, computer scientists, image-based bio-informaticians and the National Physics Laboratory Teddington (NPL), to establish and develop platforms for SRI.

The facility will be centred on commercial off-the-shelf SRI instruments that will provide a benchmark against which UCL and NPL members of the programme will develop new systems and instruments optimised for biomedical research. The facility will also provide capacity-building opportunities for students and postdoctoral fellows to train/work at the interface of biology, imaging and instrument development. Together this presents a timely window of opportunity in which to build on UCL's excellent imaging facilities and world leading biomedical research community to create a cutting edge, interactive SRI research and development programme for application to biomedical research.

We aim to develop a super-resolution fluorescence imaging (SRFI) facility that will link MRC scientists in the Cell Biology and Prion Units, as well as the MRC/UCL Centre in Molecular Medical Virology, with members of UCL's Faculties of Life Sciences, Medicine and Brain Sciences, the London Centre for Nanotechnology, UCL physicists and chemists, computer scientists, image-based bio-informaticians and the National Physics Laboratory, Teddington (NPL).

The facility will house commercial SRFI instruments for use on a range of projects with particular emphasis on cell biology, neurobiology and infectious disease. These instruments will provide state of the art bench-marks against which UCL and NPL researchers will develop new instruments, optimised for biomedical research.

We are requesting funds to:-
1. Purchase a Zeiss Elyra PS1 that will provide PALM/STORM and SIM capability, providing a multi-user platform through which UCL (Lowe, Kittler) and NPL (Knight) PALM/STORM systems and the NPL SIM system can be developed, compared and optimised. We will use UCL funds to buy a second commercial instrument to offer a broad range of imaging options.

2. Support two postdoctoral RAs to train/work at the interface of biology, imaging and instrument development under the guidance of the facility's Management Board. There will be additional capacity-building opportunities linked to the facility for UCL students and postdoctoral fellows.

3. Support development, including cloning the NPL and Lowe lab. instruments for use by UCL researchers. Significantly, the software for these instruments will be open source, allowing better assessment and control over data processing/analysis.

The facility will enable research into fundamental questions in biomedical research, providing new insights to disease mechanisms and opportunities for therapeutic development. The facility and its Management Board will also provide a focus to coordinate imaging developments at UCL.

Dissemination of findings to the scientific community.
The main route for disseminating our results will be through publication in international peer-reviewed scientific journals. Most of the project leaders in the application are leaders in their fields and are frequently invited to present their work at international meetings. In addition, user groups that meet regularly at UCL, such as the annual LMCB hosted UK Membrane Trafficking meeting, with 300 plus participants, offer ideal forums to update the national community on advances and opportunities. Thus, the results emerging from our work will have impact far beyond our immediate circle of specialists.

Dissemination of findings to the end user.
Our results and their implications will be presented to the broader scientific community and the lay public through multiple routes. We will establish a web site for the Advanced BioImaging Partnership (ABP) through which information on resources, applications, etc. will be made available. New results and tools, including open source software developed by the programme, will also be available through this site. When appropriate, information will be released through the UCL or MRC Press Offices.
The LMCB operates a schools engagement programme to encourage younger students to develop their interest in cell biology. Students will be able to visit the facility, see the instruments in use and view new findings. The facility will also be open to visitors on LMCB open days.

Establish collaborations within the scientific community and with industry.
The ABP will link biomedical researchers with instrument developers to provide a more integrated approach to SRFI. It is our intention to hold regular (at 3-6 month intervals) user meetings for results to be presented and discussed and problems resolved. We aim to create a long lasting forum through which these exciting new technologies are effectively applied and developed within UCL and extended to other similar groups across the UK. We also advocate at least annual meetings between all the advanced imaging hubs funded by the MRC, as well as links between their web sites to allow users to obtain a clear view of what is available at different locations.
The development work to be undertaken may lead to opportunities for commercialisation. Such opportunities will be taken forward through UCL Business or MRC-T.
The results from our work will be of broad interest. These new insights to the molecular organisation and function of cells will reveal new opportunities for therapy and drug development. In this respect the ABP will complement the recently established CBU/LMCB high throughput, high content screening platform.

Training of people.
The ABP will have a strong training element. UCL hosts a number of Postgraduate Training Programmes including the CBU/LMCB four year graduate programme, the new BBSRC London Interdisciplinary Biosciences PhD Consortium and CoMPLEX. CoMPLEX, provides a training programme that links physics/maths and engineering with biology. The new facility will provide opportunities for CoMPLEX students to gain insight into advanced imaging methodologies with the view that they may opt to work in this area for rotations and/or thesis projects, providing further opportunities for capacity building and development. The programme will also support postdoctoral training.

Fostering a collaborative environment.
Biomedical Imaging at UCL is well developed and focussed on the Centre for Advanced Biomedical Imaging. However, imaging at the cell and molecular level has not been as well integrated. The ABP will create a opportunity to focus this latter community by providing state of the art imaging platforms, along with seed funding and meeting forums, to encourage interactions. We believe that ABP will lead to cross disciplinary collaborations, that will make significant new insights to the molecular mechanisms of normal and abnormal cell function.