Probing cell and tissue dynamics with lightsheet microscopy

Lead Research Organisation: University of Oxford
Department Name: Physiology Anatomy and Genetics

Abstract

Imaging dynamic process in living samples is key to understanding biological regulatory mechanisms, whether at the level of molecules, cells, tissues or entire organisms. A major limitation however in most forms of microscopy is the sensitivity of living specimens to damage caused by the light used to view them, limiting the resolution, contrast and duration of time-lapse imaging. Such issues with existing live-imaging approaches have led in the past decade to the development of "light sheet" microscopy, based on optical principles developed over a century ago. These microscopes illuminate the sample with a thin sheet of light, allowing one see the sample in thin slices called 'optical sections'. By optically slicing the sample at different levels, one can build a three-dimensional view of the sample where the entire image is in focus. Other existing technologies such as confocal microscopy can create such 'optical section', but due to the way they work, impose much greater light induced damage to the sample than light sheet microscopes. A further advantage of light sheet microscopes is that the sample can be imaged from different angles. Images from these different views can then be combined to give one a very high quality 3-D representation of the sample.

This revolutionary new technology has only become commercially available in the end of 2012, from Zeiss. We tested the Zeiss microscope during the two weeks it was left here in Oxford on demo and found it to be extremely well engineered and user friendly. After less than a day of training, users were able to use it entirely independently. Because of their unique architecture, one of the most challenging aspects of light sheet microscopes is sample mounting. The instrument form Zeiss was particularly impressive in this respect, allowing one to easily mount and locate the sample, as well as maintain samples under very stable conditions suitable for extended culture and imaging, making it ideal for the projects described in this proposal.

We propose to purchase a Zeiss Lightsheet Z.1 microscope for the use of the research community in Oxford and surrounding areas such as Harwell and Bristol. The microscope will be managed by and take advantage of existing expertise in Micron Oxford, an advanced bioimaging unit that has rappidly established itself as a centralised facility serving the imaging needs of several departments in the University of Oxford.

The applicants will use the Zeiss Z.1 to address: basic biological problems that underpin future developments in human health, such as how cells in the body divide or move from one place to another; how memories are established, which will inform our understanding of the cognitive decline that accompanies aging; how stem cells produce the cells in our blood and; how the heart is built and responds to injuries, which will contribute significantly to our capabilities in regenerative medicine.

Technical Summary

The grant would be used for the purchase of a Zeiss Lightsheet Z.1 microscope. Like confocal microscopes, Lightsheet microscopes provide optical sections, but have several additional advantages because the architecture of the instrument is completely different. They cause much less damage to the sample (both photothermal and photochemical) so are ideal for time-lapse imaging of live samples. They can also easily image the sample from multiple angles, so one can build a tomographic image. This allows one to image larger samples than with a standard confocal, without compromising on resolution, which is comparable to that of wide-field systems in the image plane, and higher than conventional systems in the axial plane (the different z levels). Finally, they are very fast, collecting at over 30 frames per second.

The Zeiss Z.1 is the first commercially available light sheet microscope and has generated a great deal of excitement in the imaging community. We tested the Z.1 extensively and its capabilities in live cell imaging and fluorescence tomography compared with a leading wide field instrument (DeltaVIsion from GE), a leading spinning disc microscope (Ultraview from Perkin Elmer) and confocal laser scanning instruments from Zeiss, Leica and Olympus. The Z.1 has clear advantages for a number of key experiments in living cells, both in terms of maintaining sample viability, since the light sheet causes considerably less bleaching and photo damage than all the other modalities, but also in its ability to carry out tomography on intact tissues. We would use the Z.1 to address basic biological problems that underpin future developments in human health, are relevant to our understanding of capabilities such as memory, that deteriorate with age and will drive forward our understanding of stem cell biology and capabilities in regenerative medicine.

Planned Impact

Acquisition of the Zeiss Lightsheet Z.1 microscope will advance imaging nationally and internationally, by leveraging the existing expertise at and collaborative environment within Oxford University. By managing access to this microscope through existing mechanisms developed by Micron Oxford, a centralised bioimaging unit, the microscope will be made widely available to the research community not only in Oxford, but also outside, for example the University of Bristol and MRC Harwell, fostering an environment that encourages wide-ranging multi-disciplinary interactions. This strategic vision for the future will continue to keep Oxford at the international forefront of this increasingly exciting phase of biomedical basic and translational research.

Our use of the light sheet microscope are will result in basic discoveries thats can be translated into clinical applications. This will, in the long term, contribute to benefits in human wellbeing and to the economy. Our research will therefore have an impact on the general public and commercial sector. Given the medical relevance and ethical implications of understanding developmental processes, and its impact on stem cell biology, our work will provide factual input into and therefore benefit the public discussion about the advantages and risks of stem cell therapy.

The results of this research will be conveyed to other researchers through the publication of findings in peer-reviewed journals, by reporting unpublished work at conferences and through personal communication with other scientists. Though the results will primarily be disseminated through scientific journals, attempts will be made to inform the media of results prior to publication, so that the general public is more likely to be made aware of the results. We take seriously the responsibility of scientists to engage with the lay public, to raise awareness among them of the results of publicly funded research, to openly debate ethical issues relating to our research so that public opinion may be formed in an informed manner and to take the excitement of our research to the children of today, who will be the scientists of tomorrow. For these reasons we engage through the university but also as individuals in activities aimed at the public dissemination of science.

A dedicated web page for Micron already exists, that serves not only users, by providing a space for technical information on sample preparation for microscopy etc. but also contains pages in plain english, to promote advanced imaging approaches such as light sheet microscope to the wider community beyond researchers.

This project will also train a young researcher in emerging methodologies, contributing to their career development, as well as producing an individual capable of carrying on future research in the biomedical sciences. Micron runs an annual microscopy course that has now been over subscribed for the third consecutive year. This course is taught by several of the participants on this bid and covers a variety of imaging modalities and will include a session on light sheet microscopy.

We have set up a Bioimaging Strategy Group (BSG) comprising leading academics involved in imaging research from all interested Departments within the University. This group is chaired by Jordan Raff, one of the investigators on this application. The BSG has a central role in driving optimal coordination of research into high-end microscope development and use in Oxford. It helps prioritise and make plans for future developments and specific bids for microscopy infrastructure. The impact of the light sheet microscope will be enhanced by our coordinated plans for data storage, retrieval and fast transfer between sites, which is likely to continually increase.

Publications

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Magalhães CG (2021) Characterization of embryonic surface ectoderm cell protrusions. in Developmental dynamics : an official publication of the American Association of Anatomists

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Leonavicius K (2020) Spatial protein analysis in developing tissues: a sampling-based image processing approach. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Stower MJ (2018) The Head's Tale: Anterior-Posterior Axis Formation in the Mouse Embryo. in Current topics in developmental biology

 
Title Digital Body: Convergent Pulses 
Description Young disabled and non-disabled dancers from Oxford's Parasol Project have created a new Digital Body film with Alexander Whitley Dance Company. Filmed during the COVID pandemic, their short choreographies, inspired by conversations with members of my research group, explore how movement is coordinated within living beings. The real-world movements have been captured and edited together with 3D motion graphics by artists Robin Ashurst and Abel Enklaar at Flat 12 and features music by Rival Consoles. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
Impact Interaction between members of my group and artists. 
URL https://if-oxford.com/event/digital-body/
 
Description We have developed techniques for mounting mouse embryos in a manner suitable for light sheet microscopy.
Exploitation Route Our approaches for imagine mouse embryos can be used by other.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The Srinivas group will have a stall at the Oxford Science Festival (June 2016), where they will present their research to the lay public. We will be displaying image volumes form the Zeiss Z1 light sheet microscope in our stall. UPDATED March 2017: Our stall at the OSF 2016 was very successful - please see entry under public engagement. UPDATED March 2018: We had a stall at OSF 2017 - please see entry under public engagement
Sector Communities and Social Services/Policy,Education
Impact Types Societal

 
Description Human Fertilisation & Embryology Authority, Scientific & Clinical Advance Advisory Committee
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact Provide advice on advances in science and clinical practice which are relevant to the Authority's work in the are of Human Fertilisation and Embryology
URL https://www.hfea.gov.uk/about-us/our-authority-committees-and-panels/scientific-and-clinical-advance...
 
Description Mechanisms regulating the timing of developmental events in the early mouse embryo
Amount £791,589 (GBP)
Funding ID MR/T028637/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 08/2023
 
Description Molecular mechanisms of cell fate decisions in gastrulation and early organogenesis (WT 220379/Z/20/Z)
Amount £4,000,000 (GBP)
Funding ID 220379/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2020 
End 11/2025
 
Description UK Human Developmental Biology Initiative
Amount £6,148,973 (GBP)
Funding ID 215116/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2024
 
Description Wellcome Trust Senior Investigator Award
Amount £1,760,000 (GBP)
Funding ID 103788/Z/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2015 
End 01/2020
 
Description Wellcome Trust Strategic Award
Amount £2,400,000 (GBP)
Funding ID 105031/C/14/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 10/2019
 
Description Wellcome Trust Technology Development Award
Amount £1,500,000 (GBP)
Funding ID 108438/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 10/2020
 
Title Spatial protein analysis in developing tissues: a sampling-based image processing approach 
Description Computational pipeline for estimating protein levels from fluorescent image data. The approach incorporates features to relate expression levels with information about spatial location within the sample. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Publication of the research tool 
URL https://process.innovation.ox.ac.uk/software/p/13299a/silentmark-academic/1
 
Description Antonio Scialdone - single cell approaches to understanding early mammalian development 
Organisation Helmholtz Zentrum München
Country Germany 
Sector Academic/University 
PI Contribution Expertise in early mammalian embryology
Collaborator Contribution Expertise in computational approaches to analysing developmental processes
Impact Preprints on BiorXiv Multidisciplinary, developmental and computational biology
Start Year 2017
 
Description Ruth Baker 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution Experimental work designed to understand how cells migrate int he context of epithelia in the embryo
Collaborator Contribution Mathematical modelling of cell movements in epithelia
Impact Multidisciplinary collaboration between experimental biologists (my group) and mathematical biologists (Prof. Ruth Bakers group)
Start Year 2015
 
Description Tracking cell movements. Dr Jeyan Thiyagalingam 
Organisation Science and Technologies Facilities Council (STFC)
Country United Kingdom 
Sector Public 
PI Contribution We have provided image data that has been precessed by a machine learning algorithm to increase signal/noise.
Collaborator Contribution Our partners have developed tracking algorithms that they are adapting to track the movement of cells in the crowded epithelia of the mouse embryo.
Impact Software for tracking cells
Start Year 2019
 
Title 'SilentMark' software for the automated quantification of fluorescent signal in biological material 
Description Karolis Leonavicius (a BBSRC funded DPhil student in my group) created software that uses a novel sampling based approach for the quantification of fluorescence signal in different compartments of the cell. Used on microscopy images of immnuno-fluorescence stained samples, this allows one to quantify the amount of specific proteins in various cellular compartments. Importantly, the software also allows one to relate the fluorescence intensity to the spatial landmarks with the sample, which is particularly important in developmental biology applications. 
Type Of Technology Software 
Year Produced 2016 
Impact Thie software has been used by us in our research and by a collaborator in an unrelated project. We are in the process of publishing this software so that the wider community has access to it. 
 
Title Virtual Microscopy 
Description Software for exploring and interacting with multidimensional image volumes in virtual reality. The user can view up to four channels of data, rendered in up to six different modes. The user can also section the volume data in any arbitrary plane, in addition to zooming in/out, walking around the object, turing the object around in space etc. 
Type Of Technology Software 
Year Produced 2018 
Impact We use this software in my group to explore our image volumes and arrive at insights about the spatial distribution of proteins, or of cells within tissues. We have also found that this software is very useful in outreach activities, bringing alive our research to the lay public. We have used it in approximately 6 outreach activities, in schools, and science festivals. 
 
Description 'Dynamic Origins': Dance collaboration with Anan Atoyama 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Embryo development is a dynamic and essential time that starts our lives - something we have all undergone in our mothers' wombs but generally do not think about. An embryo starts out as a single cell which divides to form many cells that together are shaped ultimately into the new-born baby. Our research focuses on understanding how cell movement are coordinated to mould embryonic form.

A major aspect of our work relies on using microscopes to image cell movements in the embryo and computer programs to visualise these data, so that we can understand how they lead to the emergence of form. However even with these modern tools, we have difficulty sometimes truly assimilating the complicated three-dimensional changes occurring over time during development.

AToU is a dance company with a strong emphasis on creating dance piece to visualise invisible and unknown aspects of human society. Our project brings researchers and AToU artists together to visualise and more importantly, experience processes that are not easily seen, through the medium of dance. As scientists, we are very interested in AToU's work to connect concepts of embryonic body formation to our physical body movement, a unique opportunity for us to understand and inform our research through our own body, while at the same time, sharing our fascination for development with the wider public.

This project will:
1. foster deep interaction between our research group and artists, to inspire the creation of performance art based on our science by Anan Atoyama of AToU.
2. conduct science and dance workshops for students at The Cheney School in which students will co-create dance pieces with AToU and scientists.
3. organise a dance performance by the students to an audience at Cheney School.
4. produce an original music score inspired by our science and Anan's work, to enhance engagement with the Cheney School students and contribute to AToU's development of performance pieces.

Through our activities, we hope to inspire changes in attitude towards movements, art and science in the students, scientists and the artists.
Year(s) Of Engagement Activity 2019,2020,2021
 
Description Cheny School Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Participation in a Science Festival organised by Cheny Secondary School. In collaboration with Prof Mathilda Mommersteeg form my department and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) we had a table at the festival where we described out work on imaging embryonic morphogenesis over a period of approximately 5 hours. The co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant.

Acting on experience gained from our previous activity (Oxford Science Festival 2016), we were able to refine our activities for this event, as well as trial two additional activities. A student in my group created an 'World' in the computer game 'Minecraft' depicting different stages of heart development, as well as a maze through a developing heart. This attracted much interest from the secondary school students who visited our table. We also had on display cultured mouse caridomyocytes that visitors could watch beating using a microscope. In addition to these, as with our last event we had: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development.
Year(s) Of Engagement Activity 2017
 
Description Oxford Science Festival 2016 
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 Participation in the Oxford Science Festival. In collaboration with two colleagues from my department (Profs. Jo Begbie and Mathilda Mommersteeg) and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) my group had a table over two full days (Saturday and Sunday) in which we described out work on studying embryonic morphogenesis. The co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant.

Items used to facilitate engagement included: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development; and moulding clay that visitors could use to make models of embryos.

Participation in this resulted in an invitation to another science festival at a local secondary school.
Year(s) Of Engagement Activity 2016
 
Description Oxford Science Festival 2017 
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 For the second consecutive year, in collaboration with two colleagues from my department (Profs. Jo Begbie and Mathilda Mommersteeg) and Micron Oxford (a Wellcome Trust funded advanced imaging unit based in the adjoining Dept of Biochemistry) my group had a table over two full days (Saturday and Sunday) in which we described out work on studying embryonic morphogenesis. As before, the co-ordination and planning of our activities was done by a postdoctoral fellow in my group Dr. Tomoko Watanabe, who was funded previously through a BBSRC grant and is currently funded through a Wellcome Trust grant. Items used to facilitate engagement included: 3D print-outs of different stages of embryonic heart development; fixed specimens of mouse, chick and zebrafish embryos; a microscope with live zebrafish embryos; a 3D printer printing models of embryos; a game we designed where children use stickers to fill in missing stages in development; and moulding clay that visitors could use to make models of embryos. This year, we had a new display item, a 'World of Minecraft' rendering of the inside of a mouse heart. This attracted much interest from a segment of the public in the age range of 8-18 year old that we have had only limited success engaging with in the past.
Year(s) Of Engagement Activity 2017