Elyra7 with Lattice SIM microscope in the Liverpool Centre for Cell Imaging (CCI), for fast imaging of living samples beyond the limit of diffraction

Bacteria or individual cells in a plant or an animal are exposed to changes in their environment (biochemical signals, temperature, mechanical forces, light variations...), and have to interpret this information to adapt and respond appropriately. However, cells in an organism do not function in isolation - they are part of a complex 3D environment. The ability to experimentally recreate such environments and visualise individual cells, and even subcellular organisation in 3D is therefore essential to study biological processes. Imaging technologies are developing fast to study individual cells in a multicellular environment and over-time. The need to visualise cellular processes from the nanometre (to elucidate what happens inside cells) to the millimetre (to elucidate how cells are organised in a tissue) scale in the least invasive manner and in real time has triggered the development of new microscopes and imaging technologies.
We propose to purchase a newly commercialised microscope (Elyra 7, from Zeiss), which allows:
1) Imaging at very small scales (nanometre), beyond the optical resolution of traditional microscopes, without damaging the sample because of high levels of light;
2) Fast imaging of the biological processes in real-time;
3) High quality imaging of 3D samples, fixed or living, without the need of specific or complex sample preparation;
4) Imaging in physiological conditions (controlled temperature, humidity, CO2, O2).

The Elyra 7 is a versatile microscope, with multiple capabilities enabling very fast imaging of fine structures in 3D. We will install it in the Liverpool Centre for Cell Imaging (CCI), an open access and shared facility with ~100 registered users /year, from academia and industry. The microscope will serve a breadth of science across the BBSRC remit. For illustration, we here briefly present below two research topics, which will benefit from it:

1. A better understanding of photosynthesis in bacteria to further engineer crops and boost their productivity
Photosynthesis is an essential biological process. During photosynthesis, phototrophs such as cyanobacteria, algae and higher plants convert solar light into chemical energy and generate oxygen necessary for animal life. A better understanding of photosynthesis is required to drive the future engineering of crop plants to increase yields. This is achieved by the elucidation of the organisation of the very efficient photosynthetic machinery in bacteria and in algae. Groups in Liverpool and York have developed molecular tools to visualise it in living bacteria or algae. They now need to be able to image the components very precisely (at nanometre range) and to measure their fast movements. They will use the Elyra 7 for fast, and high-resolution imaging of the necessary components.

2. Imaging the processes of ageing in neurons
Ageing leads to alterations in the nervous system, which to various extent impair its functions such as capacity to learn and memory. We need to elucidate the mechanisms causing the alterations in neurons, and determine if they are irreversible or not. We will investigate how ageing affects the organisation of neuronal architecture, the intracellular organelle dynamics and the intracellular protein degradation to understand their contribution to subcellular ageing. We will require very precise imaging to detect fine changes in sub-cellular structures during ageing. We use fruitfly brains as model system, which are organised in 3D, so we will need to not only see the details, but to see them in the entire brain and in real-time. The Elyra 7 with Lattice SIM microscope will allow us to capture subcellular detail and live dynamics in the complexity of the whole brain and with reduced light-induced toxicity, all being necessary to progress our work on the cell biology of neuronal ageing.

We will install a new super-resolution lattice SIM microscope (Elyra 7)in the Liverpool Centre for Cell Imaging (CCI), capable of fast and gentle imaging in 3D, beyond the diffraction limit. It will serve a range of projects within the BBSRC remit from the University of Liverpool, the Liverpool School of Tropical Medicine, Universities in the North-West and industrial partners. This investment and the proposed projects benefiting from the new instrument, directly address the 3 BBSRC research and innovation priorities: 1) "Advancing the frontiers of Bioscience discovery", specifically tackling the "understanding the rules of life" objective by facilitating projects focusing on calcium and oxygen-dependent cell signalling, intracellular processes such as mitophagy and ubiquitin-dependent protein degradation, mitotic spindle formation etc 2) "Tackling strategic challenges" with projects focusing on "Bioscience for renewable resources: photosynthetic machinery in bacteria and "Bioscience for an integrated understanding of health": neuronal ageing, immunity, bacterial resistance. 3) "Building strong foundation", by fostering collaboration, generating knowledge transfer and training the next generation of scientists to state-of-the art microscopy. The CCI is ideally placed to offer an intensive and cost-effective usage of the equipment as it is an open access facility with prior expertise advanced fluorescence microscopy and with a strong support team for optimal exploitation of the quantitative data generated.

Where and who is our user pool?
A key beneficiary of this investment in Elyra7 will be our user pool. The CCI is setup specifically for live cell imaging in control environmental conditions with capacity to image live cell cultures (2D and 3D), bacteria (including biofilms), plants (Arabidopsis), and model organisms (Chick Embryo, Zebrafish and Drosophila). Therefore, our primary user base will be academics interested in the quantitative measurement of real-time biological events in a variety of model systems. We current host an average of 100 users from across the University of Liverpool, UK and including Industry. The Technology Directorate at UoL has signed research agreements with several North-West Universities (Liverpool John Moore University, Keele University, Liverpool School of Tropical Medicine), that provide a means for them to readily access the equipment within the CCI (via a voucher system). This has already stimulated research at the partnering institutions and promoted closer collaboration and dialogue with UoL.
Economic and Societal impact
1. The biotechnology Industry has interests in developing materials with the ability to interact with and modify the behaviour of biological materials, cultures and biofilms in specific ways (project EP5). They will benefit from access to the instrument and the data generated (see also expression of interest from Unilever and the NBIC)
2. The pharmaceutical companies will take advantage of the discoveries on intracellular processes and signals, including the better understanding of the different drug transporters, which will contribute to the improvement of drug delivery and the elucidation of new potential drug targets.
3. The agro-biotechnology industry will benefit from the new discoveries related to the photosynthetic machinery for future crop engineering (project EP1).

The Technology Directorate (TD) will assist in broadening access via voucher schemes and partnerships with other HE institutions. The CCI actively seeks industrial partners by participation to industry challenge days, such as the Unilever future challenges initiative.

Training
CCI users will receive training in SIM imaging and in image analysis by a dedicated team of support staff (facility manager, image analyst and specialist technician). On top of individual bespoke training, the CCI provides yearly workshops, which offer generic and specialist training on imaging techniques and data processing, storage and analysis. These workshops are free and available to all. In 2018, we had ~100 attendees, including 40 from outside Liverpool. Zeiss has agreed to sponsor the next three annual workshops organized by the CCI, as well as additional staff training (see support letter).
Education
The PI lectures on cell imaging and microscopy to undergraduate and post-graduate students, which channels this work into an introduction to the advantages of super-resolution imaging in life science. In addition, RL and NS have a track record of teaching in international schools. Students from different programmes will be offered lab projects affiliated to the CCI, and hence we will expose young scientists to advanced technologies and interdisciplinary thinking. The CCI will provide in depth training for the young scientists using the facility. It trains annually ~10 undergraduate and 30 postgraduate students.

Outreach
Microscopy generates visually impressive outputs, which will lead to high quality publications and materials that will facilitate outreach with powerful pictures and animations. The PIs, CoIs and CCI staff have a track record of active collaborations with the Liverpool World Museum (Meet the Scientist edition 2015, 2016 and 2018), contribution to pint of science events (2018 and 2019) and presentations in local schools. The team will use these links to host events showcasing the super-resolution imaging technique and develop teaching resources explaining the resolution limit.