A Super-Resolution Microscope for use by Plant Cell Biologists, N8 partners, Durham Scientists and Collaborators.

The first studies of biological structures were by the early pioneers of microscopy, Robert Hooke and Antoni van Leeuwenhoek, in the 17th century. Robert Hooke, in 1665, was the first to introduce the term "cell" when he was viewing the "boxes" he saw in slices of cork using one of the earliest optical compound microscopes (two lenses: an objective lens and an eye piece) that he developed. He probably didn't quite realise the significance of this discovery, as it was only when it became apparent that the great majority of organisms are composed of cells that Cell Theory was born. Cell Theory, first proposed by M.J. Schleiden and Theodore Schwann in 1839, states that cells are of universal occurrence and are the basic units of an organism. This theory is still undisputed although, in those days, rivals tried.

Over 300 years of microscope improvements have led to fascinating discoveries of how cells function and now fluorescence microscopy, a form of light microscopy where objects are tagged with light emitting dyes, has become an essential tool to study the biology of the cell. Many technical developments have led to greatly improved image quality but we are still faced with a limit in ultimate resolution when using a light microscope. Based on experiments and basic principles of physics, this 'diffraction limited resolution' was calculated by Ernst Abbe and Lord Rayleigh in the late 19th century and is approximately half the wavelength of the light being used. However, much of the fundamental biology of the cell occurs below this limit, at the level of complexes in the range of tens to few hundred nm in size; that's 10,000x smaller than a human hair.

Recently, a new generation of light microscopes, referred to as super-resolution microscopes (SRM) have been developed, which use several different methods to break through this limit. Although electron microscopes, which use beams of electrons rather than light, can magnify by hundreds of thousands of times, specimens are dead, fixed snapshots in time and require complex preparation procedures. SRM, however, can be used to look at living cells where multiple proteins or structures can be highlighted or labeled with different dyes in the same specimen.

This proposal from Durham University is requesting funds to buy one of these SRMs, in particular one which is capable of several different SR methods, so that researchers have the tools to look at a diverse array of specimens. The new equipment will be used to address important structural and cell biological questions at the nanoscale, dramatically improving our understanding of many cellular systems.

A main focus of the research using the new equipment will be on plants and crops, an area where SRM imaging has so far been limited. Specific areas which will be studied by the team in Durham include: resolving the interface between the cell's internal (cyto)skeleton with membranes a connection which is essential for cell growth; how the cytoskeleton's focus and organization changes at the site where a pathogen tries to invade the plant; how proteins and protein modifications involved in transmitting signals in the cell are arranged and their role in the plant immune response and also resolving the structures involved in the communication between the nucleus and the cytoplasm.

Importantly, the new equipment will be part of the Durham Centre for Bioimaging technology where it will be used by Durham scientists working on a range of cell systems including animal cells, fungi, alga and bacteria, also scientists that make up the UK Plant Cell Biology Community where we will share both our expertise and the facilities in order to optimize technologies for SRM, and scientists within the N8 partnership of research intensive universities in the north of England (Durham, Lancaster, Leeds, Liverpool, Manchester, Newcastle, Sheffield and York).

Current light microscopy technologies cannot resolve objects below the Abbe diffraction limit of 200nm, however much of the basic biology of the cell is at the level of complexes and structures below this limit. Several techniques, termed super-resolution microscopies (SRMs), have made it possible to image nano-environments, circumventing the diffraction barrier, yielding resolutions down to <50nm. The primary SRM methods requested here are PhotoActivated Localisation Microscopy/Stochastic Optical Reconstruction Microscopy (PALM/STORM) and 3D Structured Illumination Microscopy (3D-SIM). These techniques are based on tailored illumination or the precise localization of single molecules. In contrast to electron microscopy, Super-Resolution Microscopies (SRM) can be used on living as well as fixed tissue and multiple components can be labeled with different common dyes and fluorescent proteins.

The requested system will have a plant/crop focus and will be at the part of a UK plant imaging network that we are currently establishing with the assistance of other UK Plant Cell Biologists. The BBSRC recently supported our application to become a EuroBioimaging node. The aim of such a network would be to share both expertise and facilities in order to optimize plant imaging techniques including those for SRM and make these available to the plant community. Furthermore the new SRM facility will be part of the Durham Centre for Bioimaging Technology available for use by other members working on different cell systems, and also shared with our partners in the N8 research partnership of the eight most research intensive universities in the North of England.

Durham based projects include: the cytoskeleton and membrane Interactions; plant cell signalling, pathology and post translational modificiations; growth and development of photosynthetic organisms for Industrial biofuels; nucleo-cytoplasmic communication in plants, fungi and animals and ageing in model systems.

The objectives of the impact plan are (1) to ensure that the UK plant cell biology community have robust advanced imaging capabilities available to them (2) to work with other UK plant cell biologists to optimize microscopy techniques for use with plants and crops. (3) to provide workshops on SRM and other advanced imaging capabilities particularly on its application to plants (4) to share the facility with N8 partners and other collaborators and users working on plants as well as other cell systems (5) to protect potential applicable discoveries and raise awareness with industrial sponsors (6) to provide high level training to research associates interested in nanoscale imaging (7) to integrate with the local communities via outreach.

We will engage with the principle investigators in the UK plant/crop imaging community, many of which have sent letters of support by hosting a workshop within the first year of the programme. This aim of the workshop will be to focus on the needs of the plant cell biology community, to share ideas and technologies. The intention would be to follow this initial gathering of plant leaders with a series of workshops organised for research associates over the 5 year period on specific aspects of advanced imaging techniques which will include SRM. In addition, the SRM will be advertised to the N8 partnership and a workshop organized around the needs of this community. Co-investigators on this proposal are established leaders in animal and fungal as well as plant cell biology. This workshop will also take place within the first year of the establishment of the SRM. Moreover to help advertise the facility the SRM will be included on the Departments Microscopy and Bioimaging Facility webpages and brochure, and the Durham Centre for Bioimaging technology website. An internet gateway to the SRM facility and programme of work will be hosted by DCBT. This will offer a non-technical explanation of our activities and summarise key published discoveries for the general public, media and potential industrial collaborators.

All work and promotion of the new SRM facility will be communicated through publication in research journals and engagement at conferences open to both academic and industrial scientists.

Any exploitation of Durham based research will be discussed with the PI in association with the Durham University Technology Transfer Team who provide support to staff and research students whose research outputs have commercial significance. Groups will contact the Durham University Technology Transfer Office for example: when they may require patent protection; are interested in exploring the possibility of setting up a spin-off company or are looking to identify a commercial partner for a joint business venture. To this end, we will approach and engage with appropriate companies.

The permanent Senior Experimental Officer who runs the Microscopy and Bioimaging facility will train research associates who are part of the research groups in this programme. As a result the RAs will gain experience in SRM and an appreciation of all the technologies available in the facility. Moreover, for the wider plant cell biology community and the N8 partners interested PIs and RAs will be invited to workshops on SRM capabilities and applications (1 for each group in the first year and then as interest and need demands as described above). The Department is involved with numerous outreach activities throughout the academic year and the PI and SEO in the Microscopy and Bioimaging facility gives presentations and demonstrations of our advanced imaging capabilities. These outreach activities will continue throughout the award.

The Pi and Co-Is will be responsible for the overall management of impact . Durham University has active, well established managers and offices that give significant support to Departments for outreach activities, media relations and for interactions with industry.