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

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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).
 
Description The Dragonfly microscope was installed 2.5 years ago, and is a very popular instrument in the Centre for Cell imaging (used at 80% capacity). Multiple users including post-graduate students have been trained on the microscope and it has been used so far to serve several projects including brain tumour cell migration and adhesion, calcium signalling in c elegans, bacterial infection on teeth, virus infection, bacterial membrane photosynthetic complex organisation, stem cell regenerative medicine, 3D tumour imaging, drosophila imaging...
Exploitation Route The equipment is part of a multi-user facility. It is used by more than 10 different research groups. There are therefore a range of key findings that will be used by others. In addition, we develop imaging protocols and image analysis solution to ensure that the key findings produced on the equipment are quantitative, reproducible and can be taken forward by others.
Sectors Pharmaceuticals and Medical Biotechnology

URL https://cci.liv.ac.uk/equipment_dragonfly.html
 
Description 3DBioNet: an integrated technological platform for 3D micro-tissues
Amount £626,046 (GBP)
Funding ID MR/R025762/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 02/2022
 
Description Elucidating the organisation, activity and regulation of cyanobacterial bicarbonate transporters for engineering CO2 accumulation
Amount £330,756 (GBP)
Funding ID URF\R\180030 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2018 
End 09/2021
 
Description Evaluation of novel combination drug protocols for neuroblastoma using advanced imaging in a chick embryo model
Amount £117,000 (GBP)
Organisation North West Cancer Research (NWCR) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2018 
End 10/2022
 
Description In Vitro Organ Imaging Device (IV-OID) with integrated Biosensing and Real-Time Imaging Capability: Proof-of-Principle using a Human Placental Model
Amount £150,594 (GBP)
Funding ID BB/T012056/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 08/2021
 
Description Investigation and functional validation of post-translational modifications of proteins involved in hypoxia signalling using mass-spectrometry analysis and live cell imaging
Amount £120,000 (GBP)
Organisation MRC Doctoral Training Program 
Sector Academic/University
Country United Kingdom
Start 09/2019 
End 04/2023
 
Description Signalling In Space And Time: Intracellular Cyclic AMP Dynamics In Human Vascular Smooth Muscle
Amount £446,542 (GBP)
Funding ID BB/V002767/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2020 
End 11/2023
 
Description Single cell live imaging in vivo, to understand cell activity in the context of regenerative medicine and cancer biology
Amount £11,800 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2020 
End 01/2021
 
Description Unlocking the molecular stoichiometry of functional CO2-fixing organelles for synthetic engineering
Amount £200,000 (GBP)
Funding ID RGF\EA\181061 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2019 
End 04/2021
 
Description Validation of novel biomarkers for improved risk stratification and therapy for the paediatric cancer neuroblastoma
Amount £65,000 (GBP)
Organisation Alder Hey Children's Hospital 
Sector Hospitals
Country United Kingdom
Start 09/2019 
End 09/2025
 
Title AFM 
Description atomic force microscopy imaging on biological samples 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact We have applied high-resolution AFM imaging on many biological samples. Recently we have established a hybrid AFM/confocal/TIRF microscopy for studying cell dynamics. Structural variability, coordination, and adaptation of a native photosynthetic machinery. Zhao LS, Huokko T, Wilson S, Simpson DM, Wang Q, Ruban AV, Mullineaux CW, Zhang YZ*, Lu-Ning Liu*. Nature Plants, 2020, 6(7): 869-882. DOI: 10.1038/s41477-020-0694-3. AFM imaging on amyloid morphology provides further motivation to investigate the role of oxidative stress in AMA pathogenicity. The study has led to a paper published: Davies HA, Phelan MM, Wilkinson MC, Migrino RQ, Truran S, Franco DA, Liu LN, Longmore CJ, Madine J. Oxidative stress alters morphology and toxicity of aortic medial amyloid. Biophys J, 2015, 109(11): 2363-2370. Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center. Leanne C. Miller, Longsheng Zhao, Daniel P. Canniffe, David Martin, Lu-Ning Liu*. Biochim Biophys Acta - Bioenergetics, 2020, 1861(8): 148204, DOI: 10.1016/j.bbabio.2020.148204. AFM imaging on exosome structure has revealed that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology. The study has led to a paper published: Farahani M, Rubbi C, Liu LN, Slupsky JR, Kalakonda N. CLL exosomes modulate the transcriptome and behaviour of recipient stromal cells and are selectively enriched in miR-202-3p. PLoS ONE, 2015, 10(10): e0141429. 
 
Title AFM force measurement to study protein interaction 
Description apply atomic force microscopy force measurement to study the protein unfolding and interaction of membrane protein complexes 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Using state-of-the-art Atomic Force Microscopy (AFM), we have deciphered the nanoscale structure of the photosynthetic membranes that are extracted from a purple photosynthetic bacterium (Blastochloris viridis). Additionally, we applied single-molecule force spectroscopy (SMFS) to "pull" out protein peptides from the photosynthetic complexes in their working conditions. This allowed us to monitor the stepwise unfolding process of the structural components of photosynthetic complexes and detect the mechanical forces required in the unfolding process. Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center. Leanne C. Miller, Longsheng Zhao, Daniel P. Canniffe, David Martin, Lu-Ning Liu*. Biochim Biophys Acta - Bioenergetics, 2020, 1861(8): 148204, 
URL https://www.sciencedirect.com/science/article/pii/S0005272820300542?via%3Dihub
 
Title Absolute proteomic quantification of protein assemblies 
Description use Qconcat method based on quantitative mass spectrometry to determine the stoichiometry of protein components in macromolecular complexes. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Decoding the stoichiometric composition and organisation of bacterial metabolosomes. Yang M, Simpson DM, Wenner N, Brownridge P, Harman VM, Hinton JCD, Beynon RJ, Lu-Ning Liu* Nature Communications, 2020, 11(1): 1976. DOI: 10.1038/s41467-020-15888-4. 
URL https://www.nature.com/articles/s41467-020-15888-4
 
Description Collaboration with StreamBio 
Organisation StreamBio
Country United Kingdom 
Sector Private 
PI Contribution Stem cell-based, and more general cell therapies have shown promising results across many disciplines in medicine. However, several recent clinical studies have led to disappointing results due to the fact that the fate of the transplanted cells is unknown and fundamental mechanisms underpinning their effects not understood. This project will monitor and track the interaction over time of multiple cell types transplanted in-vivo and how they interact with the host tissues. Non-invasive cell imaging techniques on model organisms amenable for imaging, will be essential to obtain real-time, quantitative, and long-term monitoring of transplanted cells and information on cell migration, distribution, viability, differentiation etc. We will use our well-established chick embryo chorioallantoic membrane (CAM) model. CAM has the ability to support the growth and maintenance of live tissue/cells and constitute an ideal bioreactor to evaluate biological processes in regenerative medicine and cancer research. As, the chick embryo is not under Home Office regulation until Embryonic day14, its use contributes to the replacement and reduction of animal use in research. Limitations of cell tracking, to date have been down to the lack of a labelling technology, with conventional fluorescent probes suffering from poor sensitivity, photobleaching and toxicity. StreamBio's Conjugated Polymer Nanoparticles (CPNs™) are highly stable, fluorescent labelling probes, immensely brighter than current technologies, and have an iron-oxide component for MRI contrast enhancing, making them multi-modal. They are taken up by cells through endocytosis and can label any cell type. Using the advanced imaging technologies at the University of Liverpool Centre for Preclinical Imaging -CPI- and Centre for Cell Imaging -CCI- for cellular and in vivo imaging; we will determine how neural undifferentiated (neural stem cells) /dedifferentiated cells (brain tumour cells) interact with the host tissue and vasculature, and how this environment impacts on their identity in terms of differentiation, survival and migratory capabilities.
Collaborator Contribution This is a new collaboration between academics in Liverpool and an SME spun out of Kings College London. StreamBio's CPNs have superior properties not seen in other reagents, and will therefore empower several lines of research including cell tracking. StreamBio will provide the Fluorescent and magnetic nanoparticles for cell labelling.
Impact Wellcome Trust Institutional Strategic Support Fund Interdisciplinary and Industry fund (see further funding)
Start Year 2019
 
Description Halloween Science event - Outreach activity with school children 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Outreach activity entitled "Spooky Science" for Halloween. 200 primary and secondary school children attended activities at the Institute of Integrative Biology. The Liu group engaged in "Magnetic Madness" activities to exhibit the principles of protein self-assembly.
Year(s) Of Engagement Activity 2018
URL https://blogandlog.wordpress.com/2019/01/25/halloween-science-at-the-institute-of-integrative-biolog...
 
Description Liverpool Cell Imaging annual workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The Centre for Cell Imaging organised a 2 day workshop focused on light microscopy techniques, imaging probes and image analysis. 90 researchers and students attended the event mainly from Liverpool and Manchester but also from other Universities in the UK. Industrial partners were invited and attended.The second day was a focused hands-on training on image analysis.
Year(s) Of Engagement Activity 2018
URL https://cci.liv.ac.uk/2018_workshop.html
 
Description Organisation of a workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact A 2 days workshop on imaging and image analysis, attended by scientists and postgraduate students from University of Liverpool and beyond as well as by company representatives (100 attendees).
Year(s) Of Engagement Activity 2020
URL https://cci.liv.ac.uk/2019_2020_workshop.html
 
Description Organisation of the UK light microscopy facility managers meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact The Facility Managers Meeting is aimed at people running or working in light microscopy facilities.

150 Delegates from UK-based microscopy facilities find out more on the latest developments in UK Bioimaging and had the opportunity to discuss some of the basic elements (funding, impact measures) of running a core facility as well as the latest technological and application developments that effect facilities and users.
Year(s) Of Engagement Activity 2019
URL https://www.rms.org.uk/discover-engage/event-calendar/lm-facility-managers-meeting-2019.html
 
Description Outreach activity 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Local school children attended a science fair prior to the screening of the Royal Institution Christmas Lecture. The theme of our exhibit was mathematics and imaging
Year(s) Of Engagement Activity 2019
 
Description Outreach activity at the Liverpool World Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Centre for Cell Imaging led an exhibition at the World Museum in Liverpool, as part of the "Meet the Scientist" scheme. The exhibit was entitled "seeing is believing". Members of the public, including children engaged in a numerous of activities around microscopy and bio-imaging.
Year(s) Of Engagement Activity 2018
URL https://www.liverpool.ac.uk/health-and-life-sciences/public-engagement/events/meet-the-scientists/
 
Description Outreach activity with school children 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Outreach activity entitled "Spooky Science" for Halloween. 200 primary and seconday school children attended activities at the Institute of Integrative Biology. The CCI team engaged in microscopy-based activities.
Year(s) Of Engagement Activity 2018
URL https://blogandlog.wordpress.com/