PALM/STORM/SIM Super Resolution Microscopy at York

A new super-resolution microscope will bring increased resolution, sensitivity and speed to support a diverse range of leading research groups at and external to York. This will allow us to look at biological processes at a resolution and with a sensitivity that is beyond the possibilities of light microscopes currently at York.

Due to the diverse requirements of a wide user base, this microscope requested needs to have a diverse range of super-resolution capabilities implemented in a cost effective manner so as not to limit its applicability and impact on the outlined research projects. This investment in super-resolution microscopy will allow us to study living processes and differentiate macromolecular complexes within the cells at the single-molecule level. The key areas of research will initially involve studies of the following:

- The underlying mechanisms that enhance photosynthesis in algae to drive the future engineering of crop plants to increase yields
- Protein cooperativity in live cells of microbial proteins involved in gene regulation with single-molecule precision
- Subcellular localisation of DNA segregation proteins in live bacteria and archaea at the level of individual DNA segregation machines in isolated cells and within biofilms
- Resolving the spatio-temporal dynamics of proteins and LPS in the bacterial outer membrane during cell growth as a means of understanding how pathogenic and non-pathogenic Gram-negative bacteria interact with biotic and abiotic surfaces
- Key steps in how bacteria replicate their genetic material with implications in health and disease
- Studying intracellular trafficking pathways used by surface membrane proteins to discover novel and conserved trafficking mechanisms and relate these findings to disease
- Investigate the receptor monomer/dimer equilibria, receptor dynamics in the plasma membrane, and changes in receptor compartmentalisation that effect malignant cell growth
- Phosphorylation control in assembly and disassembly of the kinetochore, how the kinetochore form on the centromere, and the timing of protein complex formation as relates to the neglected disease Leishmaniasis.

At York we have always focussed on making new technologies and applications available to as many projects as possible. The microscope will be located in the Imaging and Cytometry labs in the Bioscience Technology Facility (BTF), which has an international reputation for providing expertise, instruments and training with an open access policy. As an open access facility, we have many users from outside of the University of York itself. The technology will therefore be available for use by anyone with a need to do bio-imaging with improved imaging resolution, sensitivity, or speed. With proven expert staff and supporting infrastructure, it is possible to set-up more difficult sample types for analysis and support their up and downstream analyses within the all-encompassing BTF.

In summary, by bringing a super-resolution microscope to York, we will be able to take significant steps forward in our understanding of a broad range of previously intractable biological questions, and directly underpin £27.4 million of biological funding, plus PhD-level research, with the capacity to support new interdisciplinary research initiatives in the future.

We will establish Super-Resolution Microscopy at York to support our diverse and internationally important research groups as well as opening up access to external researchers and businesses. Despite covering most resolution scales from MRI down to structural EM, X-ray crystallography and atomic force microscopy, there is a 20-100 nm resolution gap for molecular and cell biology imaging. Internal users have either been unable to gain or had limited access to external systems, which has now become prohibitive to their need to characterise biological nanostructures and biomolecular interactions. A local solution is now essential to support the internationally renowned York-based and external researchers, with the increased demand now making the investment cost effective and sustainable.

We are seeking a microscope that has two (3D SIM) to ten-fold (PALM/STORM) higher resolution, and critically for live cell imaging, has increased speed and sensitivity through 3D SIM compared to standard confocal microscopy and exceeds the capabilities of Airyscan. The system needs to be capable of a breadth of SRM methods, including range of illumination methods (epi, TIRF, HILO, PALM/STORM, 3D SIM). Multiple excitation wavelengths are required to cope with a wide range of fluorescent proteins and stains from the outlined projects. 3D SIM and 2 cameras are required to enable fast 3D tracking of organelles and screening, whilst PALM/STORM and 2 cameras are required for fast 2 colour single-molecule localisation, tracking and FRET studies. Live cell incubation, precise stage movements, with the ability to cope with multi-well plates and pre-formed channels, as well as the ability to handle microfluidics, are all needed to enhance the projects in the proposal.

The microscope will be immediately exploited by the lead groups outlined within the proposal. The microscopy lab explicitly supports ~£27.4m of current biological research grant funding, excluding PhD and other related research, with data used in ~30 publications per year from the internal user base alone.

The need for a local super-resolution microscope is now essential. This will primarily support our internal user base, whilst also being made available for external users that include other academics and commercial companies covering many remit areas of the BBSRC (e.g. tools and technologies - SteamBio and Aptamer Solutions, food productivity and health - Quorn and Cogent, improving human and animal health pharmaceutics - AstraZeneca, Tissueregenix; all of whom have ongoing York based projects) and charities (e.g. Cancer Research UK, York Against Cancer, Royal Microscopical Society - for training the next generation, York Archaeological Trust, Alzheimer's Research UK, Alzheimer's Society). All of which helps build and develop strong foundations, collaborations, partnerships and interdisciplinary pipelines through the open access infrastructure that is well proven.

As the lead groups all have current or recent BBSRC funding or closely aligned research funding, the potential impacts are already acknowledged through successful grant funding, with many other users coming from a leading multidisciplinary Department of Biology, ensuring biological impacts across the BBSRC remit.

PhD students and postdoctoral researchers will have direct access to cutting-edge equipment and will be able to apply the microscope for their own research programmes to solve important biological problems. This opportunity for training in a multi-disciplinary environment includes collaborations with industry that provides excellent career development opportunities. It will also develop biologists that will be trained in the very latest microscopy technologies and appreciate that this is not a stand-alone technique, but one that can also exploit the latest developments in genomic, proteomic and metabolomic technologies. Through our courses and events, we will also help engage a far wider audience from sponsored and paying workshops, to conference delegates and school trips. We are already incorporating new technologies into our programmes and will further incorporate new biological discoveries as the results are produced. It is amazing to see the reaction of school children and teachers alike when we show them the latest research developments; in this way we aim to inspire the scientists of the future.

In addition, the microscope itself and the biological impacts from it applications will be used as part of the microscopy-related Royal Microscopical Society and York Hands-On courses which attract ~100 delegates/year. The data will also form a natural development and progression to the external courses that we are invited to contribute to, e.g. VIB Advanced Light Microscopy (Belgium), various UK Facility Meetings, ELMI and more. All of these events are cost neutral to the grant with the running costs coming via institutional support and commercial sponsorships. These events will have both academic and non-academic beneficiaries.

With its strong visual element, microscopy is an excellent subject for outreach activities for school-age children and non-scientists demonstrating the value of research to the public and for inspiring future scientists. Demonstrations in the Imaging and Cytometry lab at York are a highlight of our school and charity visits, and 'UCAS day' tours. We also engage in external events when possible (e.g. the 'Grand Tour' which brought together artists and scientists to display scientific images in the streets with simple, engaging text). In addition, the York Communications Office is active in publicising exciting breakthroughs to the public through the university website, publications and press releases.