Fluorescence Light Sheet Microscopy for Live 3D and 4D imaging

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, light variations...). Cells have to interpret this information to adapt and respond appropriately. To understand the molecular mechanisms leading to a particular response (e.g., cell death, cell growth, cell migration, etc), biologists have to measure the levels and localisation of proteins in the cells. Each individual cell might respond differently from its neighbour and at a different time so it is crucial to follow the events in real time and in each individual cell. This can be achieved using live cell imaging coupled with the use of fluorescent labels. However, technological limitations have restricted the measurements to flat samples, such as cells attached to a glass coverslip.
In 2004, a German group invented a new microscope called light sheet microscopy, which illuminates the sample using, as the name indicates, a sheet of light. This technology also includes rotation of the sample to acquire different views. Images can be acquired very quickly through the depth of a sample (up to 1 mm thick, which is far more than with conventional microscopes). Because of the illumination geometry, the sample does not suffer from the toxicity induced by the laser light, enabling longer term imaging of living organisms. For example, the development of a zebrafish or drosophila embryo, from a few cells up to a whole organism, with single cell resolution can be observed in real-time. The first commercial light sheet microscope was released at the end of 2012. We propose to purchase one of the first commercial light sheet instruments in the UK and to install it in the Liverpool Centre for Cell Imaging (CCI). The CCI is an open access facility, so the microscope will be accessible to groups from several universities and companies. To illustrate the breadth of the science that will be served by this equipment, we briefly present below three exemplar projects:
1. Repair of muscles during ageing.
As people age, their skeletal muscles starts to deteriorate. Skeletal muscle cells do not divide and hence need to have a robust mechanism in place in case of damage or loss. Repair requires a unique population of specific muscle stem cells called satellite cells. Failure in satellite cell function can lead to delayed, impaired or failed recovery after injury and such failures increase in old age. With the new microscope, we will follow for the first time, the movement, division and differentiation into muscle cells of the satellite cells, in real time on damaged muscle fibres.
2. 3D cell culture models for drug testing
The reduction, replacement, and refinement of animal experiments is one of the BBSRC priorities. It requires better in vitro models to improve drug discovery and drug testing. Three dimensional multi-cellular spheroid models allow faster and less expensive screening in a 3D cellular organisation. To develop drug delivery and toxicity testing using 3D cell culture system, we need to understand how the cells survive, divide and move at different positions within the spheroid. Using Light sheet microscopy, we will image in real time cell survival, proliferation and migration of individual cells in the whole spheroid. This has attracted the attention of biotech companies developing biomaterials for 3D culture and pharmaceutical companies for drug testing.
3. Uncovering cell specific changes in the plant circadian clock
This project is about understanding the regulation of intracellular signals in plants triggered by the day and light changes (circadian clock). Using 3D analysis of seedlings with the light sheet microscope, we will elucidate each cell type specific regulation of the circadian clock intracellular network. This research has important implications, because the circadian clock regulates many agronomic important processes including yield, water use efficiency, disease resistance and flowering time

Technical Summary

Cell biology has undergone a revolution from largely non-quantitative observations in fixed cells to high-throughput quantitative data in live cells. Although confocal microscopy is extremely powerful for following events in single living cells, information from thicker samples is more challenging. Two photon microscopy allows deeper penetration but is limited by phototoxicity and photobleaching. Light Sheet Fluorescence Microscopy (LSFM) is a revolutionary development in microscopy. LSFM reduces photobleaching and phototoxic effects in living samples allowing long-term imaging in real time in three and four dimensions. The first description of LSFM was over a century ago, yet it was only in 2004 that Stelzer's group applied it to a developmental biology project and made the technology adequate for imaging living 3D biological samples. The imaging community has adopted LSFM and it is now commercially available (Nov. 2012 from Zeiss). At the time of writing there are no commercial LSFM systems in the UK. We propose to install a Z.1 LSFM in Liverpool Centre for Cell Imaging to serve a range of projects within the BBSRC remit for users from the University of Liverpool, as well as external academic and industrial partners. The proposed projects include imaging of circadian clock proteins in Arabidopsis, neuron mapping in zebrafish brain, cell migration in drosophila, 3D cell culture system characterisation for their use in drug delivery and toxicity, repair and maintenance of skeletal muscle during ageing, stem cell potential by re-implantation in mouse kidneys, etc. Liverpool is uniquely placed to implement this step-changing technology, by having a facility manager who was previously trained in the Stelzer's laboratory and a multidisciplinary team of developmental, cell and plant biologists as well as physicists and mathematicians for optimal exploitation of the quantitative data generated.

Planned Impact

Where and who is our user pool?
A key beneficiary of this investment in Light sheet microscopy 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, plants (Arabidopsis), Chick Embryo and Drosophila. Therefore our primary user base will be academics interested in the quantitative measurement of real-time biological events in a variety of 3D systems. We current host an average of 35 users from across the University of Liverpool, UK and the rest of the world. With recent investment from the Optical Microscopy across council initiative (MR/K015931/1) we aim to extend this. Moreover, we currently have an expression of interested with Euro-Bioimaging to become a Euro-Bioimaging Node. While our facility is well established we currently do not have any LSFM capacity.
1. The Biotechnology Industry will have specific interests in 3D structures of biological materials, cultures and biofilms. The agro-biotechnology industry will also be interested in 3D imaging of plants for example to understand plant development, target sites within a plant for herbicide resistance and tracking pathogen invasion in 3D.
2. The pharmaceutical industry will be interested in imaging cell organisation within model systems of tissues and to use those for drug testing by monitoring cell fate in real time in a 3D environment.
3. Scientific Software Industry- The 3D imaging data generated by the light sheet microscopes is generating a whole new set of challenges.
4. Scientific community. The major impact will come through access to the light sheet microscope to a wide user base. We will ensure the academic impact of this work through timely seminars, workshops and publications.
5. Outreach. The PIs and CoPIs have active collaborations with the Liverpool World Museum and local schools. The team will use these links to host events showcasing the 3D imaging technique and develop teaching resources.
6. A next generation of Scientists. The Centre for Cell Imaging will provide strong training of young scientists using the facility. The Centre for Cell Imaging train annually ~6 undergraduate and 10 postgraduate scientists, who will have access to the new light sheet microscope. The Centre for Cell Imaging is also active in with working with schools. For instance, sixth form projects are run within the CCI.
LSFM is set to become an important microscopy technique, access to this machine will be accompanied by training and workshops. On top of individual hand-on training, we plan a series of events and workshops on LSFM explaining its application as well as specific and specialist training on imaging techniques from Zeiss and external speakers. We will also include training on image analysis. Workshops will be available to new and existing CCI users in the UK.

Publications

10 25 50
 
Title collaboration with music composer Emily and launch of PRISM 
Description PRiSM is the RNCM Centre for Practice & Research in Science & Music PRiSM brings together a number of creative collaborations between the sciences and music. PRisM was launched the 04/10/2017 with "The Music of Proof" performance 
Type Of Art Performance (Music, Dance, Drama, etc) 
Year Produced 2017 
Impact Dr Howard music composition has been influenced by discussion with mathematicians and biologists 
URL https://www.rncm.ac.uk/research/research-centres-rncm/prism/
 
Description The equipment is part is a shared facility and as such is fully booked by a range of research groups and serve a broad variety of projects. Among the main developments the instrument has been used to test drug toxicity and drug delivery in organoids, to determine the function of stem cells for kidney regeneration, to test drugs targeting cancer cell migration and to test new 3D cell culture scaffolds.
Exploitation Route Lightsheet imaging provides stunning images of 3D samples, which can be quantified. We have established protocols for sample preparation and workflows for 3D image data analysis and published them in a book chapter for the light sheet community. We are also testing different 3D cell culture scaffolds and hydrogels, which will be useful for the cell biology community.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

URL http://cci.liv.ac.uk/equipment_ls.html
 
Description The equipment funded by this award is placed in the centre for cell imaging, a shared research facility at the university of Liverpool. It is currently used by more than 10 research groups, including industrial partners. It has been installed 5 years ago and has allowed to image many 3D multicellular organisation, such as multicellular spheroids, fruitflies, mouse kidney rudiments, Arabidopsis plants, synthetic scaffolds, human cornea and tsetse larvae. Mains findings are on cell invasion in a 3D matrix, pharmacological toxicity testing of compounds, and regenerative medicine. Several publications are currently in the pipeline. We organise yearly imaging workshop, sponsored by Carl Zeiss, with sessions dedicated to light sheet microscopy. They attract ~100 delegates.
First Year Of Impact 2014
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
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 Academic/University
Country United Kingdom
Start 10/2018 
End 02/2022
 
Description A Dragonfly multimodal fast imaging platform with SRRF-stream (Super-Resolution Radial Fluctuation) in the Liverpool Centre for Cell Imaging (CCI)
Amount £450,000 (GBP)
Funding ID BB/R01390X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2018 
End 04/2019
 
Description BBSRC DTP CASE studentship
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description DTP studentship
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description Development of New Mathematical Sciences for Healthcare Technologies
Amount £6,200,000 (GBP)
Funding ID EP/N014499/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 12/2015 
End 11/2019
 
Description DiMEN MRC DTP studentship
Amount £75,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 03/2019
 
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 01/2020
 
Description MRC Clinical Research Capabilities and Technologies Initiative
Amount £5,000,000 (GBP)
Funding ID MR/M009114/1 
Organisation Medical Research Council (MRC) 
Department MRC Stratified Medicine
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description clinical-academic fellowship
Amount £50,000 (GBP)
Organisation British Association of Paediatric Surgeons 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2017 
End 12/2017
 
Title Zegami 
Description A front-end visualisation platform for querying and interrogating imaging datasets. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact We have implemented a zegami server to act as a front-end to our public-facing OMERO gallery. This platform allows for the easy visualisation and interrogation of large imaging datasets and so provides an added layer of functionality. Several projects have utilised this system for Open-Data research projects, for an example see: https://doi.org/10.14293/s2199-1006.1.sor-chem.az1mju.v2 and corresponding data available at http://zegami.liv.ac.uk/2016-sf.html 
URL http://zegami.liv.ac.uk
 
Description Aurelia Bioscience 
Organisation Aurelia Bioscience
Country United Kingdom 
Sector Private 
PI Contribution Aurelia Bioscience is a research contract organisation. Their main source of revenues is large scale testing of compounds for toxicity or efficacy for the pharmaceutical companies. They are interested in 3D models as these might be more representative of in vivo systems. With the light sheet microscope, we have ran test experiments to demonstrate the possibility of visualising the fate of cells growing on the 3D scaffolds Aurelia Bioscience is developing. The very encouraging results have led to a successful BBSRC CASE application.
Collaborator Contribution Aurelia Bioscience procured the 3D scaffolds and grew various cell types. They came to Liverpool Centre for Cell Imaging to do the experiments with us. They are committing some additional funding through the CASE application.
Impact BBSRC Case Studentship to start October 2016 (Newcastle-Liverpool-Durham BBSRC Doctoral Training Partnership) The collaboration is highly multidisciplinary, involving physics, mathematics, data processing, biology, material chemistry and pharmacology.
Start Year 2015
 
Description Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We develop long term imaging and superresolution imaging in an oxygen controlled environment
Collaborator Contribution Zeiss has provided training and support for imaging technology development
Impact Yearly imaging workshops and one BBSRC DTP case studentship
Start Year 2013
 
Description workshop focused on cell imaging and image analysis 
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 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 the director of the Advanced Imaging Centre at the HHMI Janelia Research Campus, Teng-Leong Chew gave a keynote lecture and a comprehensive hands-on training session on Image Analysis.
Year(s) Of Engagement Activity 2017
URL https://cci.liv.ac.uk/2017_workshop.html
 
Description "Meet The Scientist" exhbit at the World Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Our exhibit was entitled: "Seeing inside cells". More than 300 visitors stopped at our exhibit and learned about cells, and how we can use microscopy to understand how they function. Children have visualised cells using a fluorescent microscope.
Year(s) Of Engagement Activity 2015
URL https://www.liverpool.ac.uk/health-and-life-sciences/meet-the-scientists/21-november/
 
Description Hosting Lower 6th form summer students via the Nuffield scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The Centre for Cell imaging has hosted 2 6th form students for 4 weeks as part of the Nuffield Foundation scheme
Year(s) Of Engagement Activity 2017
 
Description Image Analysis Blog 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact A blog, outreach and teaching resource based around the work done by Dave Mason, the CCI BioImage Analyst. The tutorials and educational material largely focuses on Open Source and Open Access tools such that others can apply the methods and techniques to their own quantification.

This platform also provides a route to engage with the wider community through comments and interactions on-site and via other social media platforms (IE twitter).
Year(s) Of Engagement Activity 2015,2016,2017
URL http://postaquisition.wordpress.com
 
Description Imaging workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact More than 50 attendees participate to the yearly workshop organised by the Centre for Cell imaging. it provides training and awareness of new imaging technologies to group leaders, members of staff and students from the University of Liverpool, other Universities and local companies.
Year(s) Of Engagement Activity 2012,2013,2014,2015
URL http://pcwww.liv.ac.uk/~cci/2015imagingday.html
 
Description Imaging workshop September 2016 
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 Annual imaging workshop, focused on fundamentals in imaging and specific seminars on microscopy techniques such as super-resolution microscopy, intravital imaging, Life-time imaging. The main purpose is training and increasing awareness of the imaging capabilities in Liverpool
Year(s) Of Engagement Activity 2016
URL http://cci.liv.ac.uk/2016imagingday.html
 
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 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 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/
 
Description Primary school visit for the Science week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact The activity consisted in 2x2h sessions with 2 year 6 classes in a primary school in Liverpool (Mosspits Lane primary school) for the Science week 2017. The children were offered activities focusing on scales in biology, use of microscopy and building their own magnifier.
Year(s) Of Engagement Activity 2017
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I gave an introductory lecture on molecular and cellular biology of cancer and have demonstrated how 3D/4D imaging has helped our understanding of cell migration. The students were fascinated and provided excellent feedback.
Year(s) Of Engagement Activity 2015
 
Description School visit to the CCI 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 50 A-level students visited the Institute of Integrative Biology, and were given a talk about imaging in the 5 dimensions.
Year(s) Of Engagement Activity 2018
 
Description School visits 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact We have regular visits from groups from Secondary School in the Institute (in addition to the Open Days) and the visit of the Centre for Cell Imaging is always a high point as illustrated by the testimony from one of our visitor (see link below).
Year(s) Of Engagement Activity 2015,2016,2017
URL https://blogandlog.wordpress.com/2016/08/15/visit-from-a-level-biology-students/
 
Description Scientific oral presentation at the Dynamic Cell conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Oral presentation of Research data on 3D imaging of cell invasion.
Year(s) Of Engagement Activity 2018
URL https://bscb.org/meeting/dynamic-cell-iii/
 
Description Talk at EMBO symposium "Seeing is Believing- 2017" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Short talk regarding an innovative hydrogel for embedding samples for different kind of microscopy, mainly Light Sheet Fluorescence Microscopy. The talk triggered quite some interest and samples of this hydrogel were requested by several international institutions including Morgridge Institute for Research (Winsconsin, USA) and European Molecular Biology Laboratory (EMBL, Heidelberg)
Year(s) Of Engagement Activity 2017
URL https://www.embo-embl-symposia.org/symposia/2017/EES17-08/index.html