Liverpool 3View: a national hub for 3D-EM bioscience research

Lead Research Organisation: University of Liverpool
Department Name: Institute of Translational Medicine

Abstract

New 3View equipment is required for imaging the fine details of the insides of cells and tissues in 3D. In all of our projects we are using multidisciplinary approaches to address our questions however they are all united by the need for imaging at high resolution. The challenge with this is that the closer you look the harder it is to see the surrounding context. The solution is to take images of many serial sections through the sample and then digitally reconstruct them into a 3D volume that can be viewed from any angle. This helps with understanding the spatial relationships between cells within tissues, between subcellular compartments and for observing rare events. The 3View system makes this type of analysis routine, allowing rapid processing of samples.
Our projects span a broad range of subjects from basic cell biology and physiology such how cells organise their communication systems when they are moving, how the recycling machinery of the cell works, changes to cells and tissues during ageing and the biogenesis and structural organisation of healthy tissues. Our work also extend through to translational projects, for example interactions between engineered stem cells and host tissue, development of novel cell scaffolds for treating age-related eye disorders, infection routes taken by parasites or bacteria in the gut and the development of novel nanoparticle tools for delivering cargoes to cells.
The equipment will be housed in a leading UK electron microscopy unit and available to external scientists from the UK and Europe. 3View is a new technology that is only beginning to become established within the UK. As part of making Liverpool a national hub for this technology and to help drive innovative applications, we will set up a workshop programme that will act as a forum for experienced users and will enable new users to learn about sample processing, imaging and analysis.

Technical Summary

We would like to purchase a Gatan 3View2XP system to enable high-resolution 3D-electron microscopy (EM) of cell and tissue volumes to be routinely performed. Our proposals will improve understanding of: i) cell signalling integration and compartmentalisation; ii) organelle biogenesis, structure and interactions; iii) aging related changes in model organisms and human tissue; iv) stem cell engineering for translational applications; v) host-parasite and gut-bacteria interactions required for infection; vi) cell-matrix and musculoskeletal biology in humans and animals; vii) cellular interactions of novel biofunctionalised nanoparticle reagents.
The projects described represent multidisciplinary approaches to address the research questions. The common theme within all of the projects is the need for 3-D EM imaging of cell and tissue volumes to allow visualisation of subcellular compartments, cell-cell and cell-substrate interactions, tissue morphology and organism anatomy. The proposal incorporates a consortium of local and external academics from leading UK research institutes with immediate requirements for access to this new technology. Since we are a leading bio-EM unit within the UK with a critical mass of high quality projects, we are well positioned to extend our capabilities to become one of the national hubs for this technology with reserved capacity for external groups.

Planned Impact

The proposed projects will impact the following groups over the indicated timescales
- Local and external researchers that will access the equipment (immediate impact)
- Scientists within & outside research field of the PIs (months, years: Detailed in Academic Beneficiaries)
- Medical and veterinary clinicians (months, years)
- Biotechnology companies (years)
- General public (months, years)

3View is a new technology that was initially developed and established in Germany followed by a rapid uptake of installed capacity in the US and the Far East. The UK has an international reputation in advanced imaging capabilities and research and 3View is highly complementary to many of these approaches. However, we currently lack national 3View capacity to serve existing demand with consequent risks to scientific competitiveness. A strategic investment in this technology to improve UK capacity has the potential to benefit any groups that use imaging in their research. There will be a specific benefit to BBSRC-funded researchers. All current 3View systems have been funded by the MRC, the Wellcome Trust or CRUK. We propose to reserve a minimum of 20% of our capacity for external users with priority given to those with BBSRC funded projects.
All users of the equipment will be offered full training in advanced skills of sample preparation, imaging and image processing. We will also establish a workshop that will provide a national focal point for new and experienced users. The workshop will involve leaders in 3View imaging from the UK and Europe and facilitate training, discussion of best practice, troubleshooting and provide networking opportunities. Together, these initiatives will contribute to the professional development of all users of our facility and help to promote co-operation amongst the network of 3View facilities within the UK.
Scientifically, our application is focussed on our strengths in cell biology, physiology, biomedical and veterinary research. The work that will use the 3View includes highly translational studies, for example, the investigations into bacterial-gut interactions and the role of dietary factors at modulating this (Campbell). Advances in this area have potential human quality of life and health benefits and may inform government policy and industry protocols for example in the types of additives used in food. Similarly, the work that will be done to understand the mechanisms of invasion of Trypanosomes across the Tsetse fly gut wall (Acosta-Serrano) has the potential to reveal new therapeutic avenues over the next decade for treating this debilitating disease. Research into age-related deterioration of tendon mechanical function (Laird & Clegg) may provide insights into methods of limiting injury risk that will inform treatment practices and drug development. Other examples with potential to have medical benefits and biotechnology company involvement include: novel nanotechnologies for drug delivery (Brust and Jones), developing new stem cell tools for treating kidney disease (Wilm & Murray) and transplants for treating age-related eye disorders (Williams).
The Biomedical EM unit and all participating co-applicants are committed to engaging with the public about their research. These include visits to the facility and labs by schools, fundraisers and patients; producing websites written in a lay style, publicising the results of our research via local and national press with support from funding agency/University press offices and participating in educational forums.
 
Description This capacity building award has meant that we have successfully introduced 3View 3D-EM capabilities to Liverpool. This has allowed a wide portfolio of local and national scientist to access this resource to address questions related to cell and tissue organisation and physiology. Preliminary findings are likely to be used for further grant applications from the client scientific groups.
Exploitation Route Work in the EM facility using the 3View is necessarily varied. For example, groups from tropical medicine are investigating malaria parasite infiltration of mosquito guts that may lead to novel treatment opportunities. Novel organisational features of the endoplasmic reticulum in pancreas that have been identified will inform basic research into calcium signalling and membrane trafficking (Dickens et al, 2016). Finally, we recently performed the first ultrastructural analysis of the complete human genome and found that chromatin contributes a smaller fraction to chromosomal volume than expected (Booth et al, 2016). The relocalisation of a novel chromosomal peripheral compartment was characterised and will inform future research studies.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The funding was used to purchase a 3View 3D-EM system for ultrastructural research. The equipment was embedded into an established electron microscopy facility with a broad client based from bioscience, biomedical, chemistry and engineering disciplines both locally in Liverpool and nationally. The equipment is fully functioning and has been generating preliminary data that research groups are intending to use for full grant applications. Furthermore, we are refining methodologies that will improve the range of samples that can be used in this application.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description 3D-EM - Cambridge 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Public 
PI Contribution We have performed all of the EM/3D-EM related work to investigate the organisation of the endoplasmic reticulum in relation to other organelles
Collaborator Contribution Collaborators direct the research project and are responsible for all non-EM related work.
Impact Dickens JA, Ordónez A, Chambers JE, Beckett AJ, Patel V, Malzer E, Dominicus CS, Bradley J, Peden AA, Prior IA, Lomas DA and Marciniak SJ. The endoplasmic reticulum remains functionally connected by vesicular transport following its fragmentation in cells expressing Z-a1-antitrypsin. FASEB J (2016) 30:4083-4097.
Start Year 2015
 
Description 3D-EM - Edinburgh 
Organisation University of Edinburgh
Department Centre for Integrative Physiology
Country United Kingdom 
Sector Academic/University 
PI Contribution We are collaborating with Professor Bill Earnshaw and other colleagues at Edinburgh to provide 3D-EM imaging of samples addressing questions relating to DNA organisation, cell cycle and cytoskeletal organisation. We are responsible for the EM imaging whilst our collaborators provide the biological samples and direct the overall research projects.
Collaborator Contribution Our collaborators lead the research projects and perform all non-EM related works.
Impact Booth DG, Beckett AJ, Molina O, Samejima I, Masumoto H, Kouprina N, Larionov V, Prior IA and Earnshaw WC. 3D-CLEM reveals that a major portion of mitotic chromosomes is not chromatin. Mol. Cell (2016) 64:790-802.
Start Year 2015
 
Description 3D-EM - LSTM 
Organisation Liverpool School of Tropical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We perform all of the 3D-EM work on trypanosome/TseTse fly samples to investigate locations of parasitic invasion of the gut.
Collaborator Contribution The group of Dr Alvaro Acosta perform all of the non-EM related work and direct the research project.
Impact N/A
Start Year 2015
 
Description 3D-EM - Warwick 
Organisation University of Warwick
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We are performing all of the 3D-EM for work that is investigating the organisation of the cytoskeleton during mitosis.
Collaborator Contribution Steve Royle and his lab perform all of the non-EM related work.
Impact N/A
Start Year 2015