A Dragonfly multimodal fast imaging platform with SRRF-stream (Super-Resolution Radial Fluctuation) in the Liverpool Centre for Cell Imaging (CCI)

Individual cells in a plant or an animal are exposed to changes in their environment (biochemical signals, temperature, mechanical forces, light variations...). Cells have to interpret this information to adapt and respond appropriately. However, cells in an organism do not function in isolation, but are part of a complex 3D environment. The ability to experimentally recreate such environments and visualise individual cells in intact organs or 3D cultures is therefore essential to study biological processes. Imaging technologies are developing fast to study individual cells in a multicellular environment 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 microscope (Dragonfly, commercialised by Andor) which allows:
1) Fast imaging of the biological processes in real-time,
2) High quality imaging of 3D samples, fixed or living, without the need of specific sample preparation,
3) Imaging at very small scales, beyond the optical resolution of traditional microscopes, without damaging the sample due to high levels of light
4) Imaging in the near-infrared spectrum of light, which is more commonly used for in vivo imaging. This will enable to integrate measurements done with the same fluorescent probes from cells to tissues in small animal models.
5) Imaging in physiological conditions (controlled temperature, humidity, CO2, O2).

Dragonfly is a versatile microscope, thanks to its 3 different modes of illumination and is an ideal instrument for a multi-user facility. We will install it in the Liverpool Centre for Cell Imaging (CCI), an open access and shared facility with ~100 registered users from academia and industry. The microscope will serve a breadth of science across the BBSRC remit. We briefly present below two research topics, which will benefit from it:

1. A better understanding of photosynthesis in bacteria and algae 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 Dragonfly for fast, high-throughput and high-resolution imaging of the necessary components.
2. The unexpected role of oxygen sensing proteins in cell division
Oxygen is essential for life in multicellular organisms and animals have evolved mechanisms to cope with decreased oxygen concentration. Interestingly, oxygen-sensing proteins have recently been discovered, to also regulate essential processes during cell division. The molecular mechanisms that connect oxygen-sensing enzymes and cell division needs to be further investigated, using models of human tissues. Because cell division processes are fast and occur in defined areas of the cells, a microscope allowing high speed imaging and high resolution is required. Moreover, the pseudo-human tissues recreated in vitro for this study are fragile and require the gentle imaging conditions, with low light levels, to avoid light-induced damages. The Dragonfly will provide the required imaging conditions for this study and will enable the discovery of important mechanisms that control cell division in human tissues.

We will install in the Liverpool Centre for Cell Imaging (CCI) a new versatile microscope capable of multiple imaging modalities. The multi-point confocal provides high-speed and high-sensitivity image capture, considerably reducing phototoxicity and photobleaching during live cell imaging capture. The instrument will be equipped with super-resolution based on radial fluctuation analysis (SRRF). SRRF is capable of live-cell super-resolution imaging over timescales ranging from minutes to hours, using sample illumination orders of magnitude lower than PALM, STORM or STED. Moreover, thanks to the Borealis illumination, the microscope is suited for near infra-red imaging, a capability which is particularly important in Liverpool, to strengthen the imaging workflow from cells to animals, harnessing the combination of imaging capabilities between the CCI and the Centre for Preclinical Imaging. The platform is also perfectly suited for fast volume acquisition of 3D samples. It will replace an old confocal microscope no longer maintained by the manufacturer and it will serve a range of projects within the BBSRC remit from the University of Liverpool, York, Keele and external partners. The proposed projects directly address the 3 BBSRC strategic priorities: 1) Food security via future crop engineering with photosynthesis components from algae, 2) Bioenergy via the elucidation of the photosynthetic machinery in bacteria and 3) Bioscience for health via improved stem cell tracking for regenerative medicine, improved drug testing for efficacy using organoids and the increased understanding of key cellular processes including cell cycle, cell migration, and adaptation to low levels of oxygen. 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 in super-resolution fluctuation analysis 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 Dragonfly 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, 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 the rest of the world, including Industry. The Technology Directorate at UoL has recently signed research agreements with several North-West Universities (Liverpool John Moore University, Keele University, Liverpool School of Tropical Medicine, with more agreements due to be announced shortly) that will provide a means for them to readily access the equipment within the CCI. This will stimulate research at the partnering institutions and promote 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. They will benefit from access to the instrument and the data generated (see letter of support from Unilever)
2. The pharmaceutical companies will take advantage of organoid imaging with the Dragonfly for improved drug toxicity and efficacy screening. Professor Sir Munir Pirmohamed is a clinical pharmacologist, and the NHS Chair of Pharmacogenetics and has many contacts with a range of pharmaceutical industry partners, who will directly benefit from the new instrumentation.
3. The agro-biotechnology industry will benefit from the new discoveries related to the photosynthetic machinery for future crop engineering (projects EP1 and EP3, Case for Support).

The Technology Directorate (TD) will assist in broadening access via voucher schemes and partnerships with other HE institutions. The CCI actively seek industrial partners by participation to industry challenge days, such as the Unilever future challenges initiative. The CCI currently has 4 regular users from industry.

Training
CCI users will receive training in confocal, widefield, SRRF imaging and 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 2016, we had ~100 attendees, including 40 from outside Liverpool. Andor has agreed to sponsor the next three annual workshops organized by the CCI (see Andor 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. 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 and CoIs have a track record of active collaborations with the Liverpool World Museum (Meet the Scientist edition 2015 and 2016) and local schools. The team will use these links to host events showcasing the 3D imaging technique and develop teaching resources. The CCI also hosts 6th form students for short projects (Nuffield bursary scheme).