MICA: Bio-Continuum Microscopy: Seamless imaging from the micro- to the nano-scale

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Recent advances in biological imaging have focussed on improving resolution in both living and cryopreserved tissue. Despite exciting developments, the new techniques are still not ideal for many biological studies and remain far from simple to apply for non-specialists. To achieve a full understanding of the complex biological systems that underlie disease/infections and fundamental biology, it is imperative to understand all aspects of the mechanisms involved. Recent years have seen genomic/proteomic studies further our understanding, but we are still unable to visualise many key processes directly in an intact living cell. Thus, whilst recent developments are bringing us closer to this goal, a critical resolution gap between light and electron microscopy remains.

Our electron-excited Super Resolution Microscopy (eSRM) technique will build on our Fluorescence Electron Microscopy (FEM) approach and seamlessly couple the technologies of light and electron microscopy to achieve a paradigm shift in biological imaging. This approach brings together the unique ability to image multiple coloured tagged-particles, such as antibodies, with the resolution of the electron microscope, whilst only requiring standard light microscopy preparations. It is therefore simple and easy to use across a broad range of biological questions. Preliminary work has established proof of concept that multicolour images can be acquired with a resolution of 3 nm on cell membranes. We are now uniquely placed to develop these methods and address vital medical/biological questions.

Our ability to preserve fluorescent proteins (GFP) through sample preparation for electron imaging will revolutionise electron microscopy just as the same fluorophores did for light microscopy several decades ago. Our groundbreaking integration of SRM with both SEM and volume EM (3View) will allow us to exploit these developments quickly and efficiently to deliver accurate protein localisation in situ without complicated sample processing.

Our strategy will have a major impact on the microscopy field; it will go beyond the information gained through recent advances and enable us to undertake molecular localisations and interactions under more relevant biological conditions. Current methods have a number of limitations that curtail data interpretation. For example, they rarely detect all the labelled proteins, require extensive imaging strategies and high resolution is usually achieved through mathematical algorithms rather than directly. Our techniques (eSRM, SRM coupled with CLEM) will enable studies at physiological concentration, visualise true molecular associations/distributions and permit study of fine-detailed sub-cellular and cell surface structures and changes in response to various stimuli. A key aspect of our approach is to enable easy sample handling and labelling so that non-specialist microscopists can use the methods. Our probes will be widely applicable to many microscopy methods, from in vivo multiphoton through to light emission from electron excitation. They are more photostable than existing probes and have very narrow emission bands, making multicolour imaging simpler. They have improved sensitivity and longer lifetimes which will be exploited when the very latest technologies are launched this year.

In summary, by bridging the current gaps to produce a continuum light and electron microscopy approach and through combination of our latest advances, we aim to develop an integrated approach that far surpasses other techniques being developed. This will enable the step change required in microscopy to enable biologists to undertake true multicolour sub-light resolution imaging with very few constraints or specialist training, thus addressing a major limitation in biology and representing a ground-breaking advance in biological imaging.

Technical Summary

We will develop new imaging methods:
1 Integrate standard SRM with our novel electron-excited SRM making this technology simple to use. We will use our novel probes (see 3 below), which can be excited with both light and electron beams. Building on our expertise in standard SRM and CLEM, and our relationship with JEOL, we will improve the optical resolution of JEOL's novel ClairScope, a combined light/electron microscope. This will be achieved by adding a high performance PMT to the optical light path to enable ca 20 nm multicolour resolution. We will also build Bayesian Imaging onto the system for comparative imaging.
2 Develop integrated SRM-SEM for direct CLEM imaging. DELMIC will integrate PALM with the SECOM platform to study dual-label specimens in situ. Exploit our own recent advances in preserving fluorescent signals such that they can be used to give precise overlay of fluorescence and EM images. This will permit ca 2 nm resolution of samples. Finally we will integrate miniaturised fibre-optic laser microscopy into the automatic serial imaging workflow of the 3View platform to enable the analysis of complex networks in larger samples.
3 Further develop our novel probes. Optimise the probes for brightness and size distribution to be used in 1 and 2 (above) such that we will be able to image individual particles. We will also change the dopants so that the probes can also be used for live cell, MP imaging and then as fiducial markers for CLEM.

These developments will address key biomedical questions, focussing on mammalian cell biology and molecular/cellular microbiology including
-imaging of tissue, cellular and subcellular processes related to cancer.

-visualizing activation of silent synapses related to learning and memory.

-molecular/cellular microbiology questions related to bacterial and parasitic infections, the formation of bacterial communities and the mechanisms of bacterial chromosome segregation and plasmid partitioning.

Planned Impact

The development of multicolour high resolution imaging below the wavelength of light to address key biomedical questions will provide impact in areas beyond academia.

Companies and the UK economy.
Equipment manufacturers JEOL and DELMIC are key collaborators; both companies are significantly supporting the proposal through a combination of direct funding, provision of equipment at cost prices in addition to associated consumables and significant in-kind staff time and maintenance cover provision. Our developments will help impact their own work with regards to their new ASEM and CLEM approaches by advancing them to specifically address biomedical questions not previously envisaged. We will also be working closely with local companies who specialise in the development of aptamers. Aptamer technology is a very competitive market with multiple young companies being set up across Europe. By working with our local spin out companies in York, we will not only improve our resolution, but also enable new probes and approaches to be directly developed with the biomedical market in mind.

The project aims will benefit further non-University researchers. Obviously CRUK will be direct and immediate benefactors, but the techniques will be easily adapted by other local organisations, such as the DEFRA-funded Food and Environment Research Agency who has already collected data using the ClairScope. Large pharmaceutical companies, food manufacturers and oil companies have also expressed interest in this technology. Making super-resolution microscopy an easy to use technique (as proposed) will ensure its application in non-academic environments.

Well-trained workforce and the UK economy.
The technical staff, PhD students and postdoctoral researchers directly involved in the proposal will have the benefit of developing and using equipment at the cutting edge of biological microscopy. They will also have the benefit of participating in, and understanding the different requirements of, industrial partnerships and how scientific advances can be exploited beyond academia. Through our courses and events, we will also help engage a far wider audience from sponsored and paying workshop and conference delegates to school trips (see Impact statement). We are already incorporating the new technologies into our programmes and will further incorporate the biology as the results are produced. It is amazing to see the reaction of school children and teachers alike when we show them the latest research developments; in this way we aim to inspire the scientists of the future.

Patients and the UK economy.
The biological problems to be investigated are relevant to human health and disease. Cancer causes more than one-in-four of all deaths in the UK and more than three-quarters of deaths are for patients over 65. Cancer is not only a distressing and life-threatening disease but will become an even larger challenge for health services as the population ages. Normal synaptic function in the brain is not only a fascinating area of research in providing insight into how we learn but loss of function and memory loss are problems associated with ageing and which are particularly distressing for the families of those affected. Biofilm infections (particularly on clinical devices) affect millions of patients worldwide every year, threaten the positive impact on length and quality of life provided by prosthetic devices, are a particular problem in intensive care and neonatal units and are a very significant economic burden on health services.
The aim of all the biological research programmes included in this proposal is to use developments in probes and correlated light and electron microscopy to ask questions that were previously inaccessible. The aim is for an improved understanding of the biological processes that will lead to the identification of novel therapeutic targets for prevention and/or treatment of disease.

Publications

10 25 50
 
Title #ImagingScientist 
Description Film, images and interviews for www.imagingscientist.com website 
Type Of Art Film/Video/Animation 
Year Produced 2018 
Impact Used by UKRI, viewed internationally, helps influence opinion 
URL http://www.imagingscientist.com
 
Description BBSRC - Critical Friends of Bioimaging
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description BBSRC TRDF Grant Awarding Panale
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description BioImaging UK
Geographic Reach Europe 
Policy Influence Type Citation in other policy documents
 
Description Microscopy Courses 2013-2020 onwards
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Idea to Innovation Award
Amount £15,000 (GBP)
Organisation Francis Crick Institute 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 07/2017
 
Description Proximity to Discovery: Industry Engagement Fund
Amount £25,055 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 08/2018
 
Title UltraLM 
Description The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Publication of tool for use by correlative imaging community. 
URL https://wellcomeopenresearch.org/articles/1-26/v1
 
Title miniLM 
Description The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Publication of tool to enable build and use by correlative 3D EM imaging community. 
URL https://wellcomeopenresearch.org/articles/1-26/v1
 
Description Access to SBF SEM 
Organisation Amherst College
Country United States 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Babraham Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Curie Institute Paris (Institut Curie)
Country France 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Imperial College London
Department Faculty of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Leiden University Medical Center
Department Molecular Cell Biology (LUMC-C)
Country Netherlands 
Sector Hospitals 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation Open University
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology (LMCB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation University of Aberdeen
Department Institute of Medical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation University of Bristol
Department School of Biochemistry Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Access to SBF SEM 
Organisation University of Stellenbosch
Department Department of Physiological Sciences
Country South Africa 
Sector Academic/University 
PI Contribution Access to our Serial Block Face SEM for 3D EM of cells and tissues. Open access collaborations delivered as stated in MRC grant.
Collaborator Contribution Primary biomedical research and associated publications.
Impact Publications as follows... 26584158 26181626 25257211 28139674 27510922 27445312 27338237 27009270
Start Year 2013
 
Description Zeiss Labs at Location 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution The increased exposure of York through the grant funding has enabled us to open up space and resource to become a flagship demonstration site for their instruments. This has included beta-testing and now an active demonstration site. We will provide the infrastructure and expertise.
Collaborator Contribution They have loaned, free of charge, a top end light sheet microscope which will be openly accessible to any academic parties both inside and outside of York and the consortium. This will enable anyone outside of York to gather proof of concept data whilst York academic groups get free access to a ~£350,000 microscope which we would not otherwise have in-house.
Impact Just started!
Start Year 2014
 
Title 3D SR ILSEM 
Description 3D super-resolution integrated light and electron microscopy - an advanced tool for accurate ultrastructural localisation of fluorescently-tagged macromolecules in cells and tissues. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact Manuscript in preparation. 
 
Title MiniLM 
Description Miniaturised light microscope for integration into ultramicrotomes and electron microscopes - a tool for localisation of fluorescently-labelled regions of interest for further ultrastructural analysis. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact Manuscript in preparation. 
 
Description Article on the Francis Crick Institute Electron Microscopy Science Technology Platform for In Focus Magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Article on the Francis Crick Institute Electron Microscopy Science Technology Platform for In Focus Magazine
Year(s) Of Engagement Activity 2017
URL https://www.rms.org.uk/study-read/infocus-magazine.html
 
Description Crick Chat on Electron Microscopy: Seeing the Invisible 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact In the very first Crick Chat to be held inside the new building, Lucy Collinson, who leads the electron microscopy science technology platform at the Crick, and Gipi Schiavo, a neuroscientist from UCL's Institute of Neurology, discussed how advances in electron microscopy are allowing us to see inside cells in minuscule detail, and what this means for treating disease. Crick Chats are relaxed and friendly evening events that bring the general public face-to-face with researchers working at the Francis Crick Institute and our partner organisations. Hear about their work, ask a burning question, or just enjoy a chat; Crick Chats are free and open to all. No knowledge of science is required. Discussions were held with several patients undergoing treatment for neurological conditions, as well as further meetings organised with a retired specialist in scientific quality assurance, and a researcher studying collaboration netwroks in the newly formed institute.
Year(s) Of Engagement Activity 2016
URL https://www.crick.ac.uk/whats-on/public-events/catch-up/
 
Description Exhibit at Royal Engineers Summer Soire 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Over 100 Royal engineers and guests, plus Princess Ann and others

Article in the Financial Times used our research quotes and images relating to the MRC funded grant
Year(s) Of Engagement Activity 2013
 
Description Guestbook article for Laboratory News 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Guestbook article for Laboratory News on correlative microscopy and big data
Year(s) Of Engagement Activity 2017
URL https://www.labnews.co.uk/interviews/guestbook/microscopy-always-stronger-together-28-06-2017/
 
Description Newspaper interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Article in the Financial Time Weekend Magazine

Increased publci awareness
Year(s) Of Engagement Activity 2013
 
Description School visits (Local York Schools, e.g. Fulford, The Mount) 
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 We run live and partially interactive demonstrations of the microscope facilities which includes the technologies used and being developed within these funded projects. They are in small groups to permit close interaction and walk them through multiple repeated workstations throughout a 2 hour programme.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Science magazine interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact An extensive 2 page article about the MRC funding and research being undertaken here at York and with our collaborators at the LRI, CRUK

Distributed as part of a Micorscopy Special edition
Year(s) Of Engagement Activity 2013
 
Description Seminar on International Hands-on Confocal Microscopy Course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact This seminar is given to the advanced delegates on the confocal microscopy course demonstrating the further applications that are available. This also includes tours of the facilities and instruments being used as part of this project.
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Talk at BioBeat17: Reshaping BioTech Partnering 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Talk at BioBeat17: Reshaping BioTech Partnering, bringing together scientists, entrepreneurs, biotechs and industry to discuss how to develop partnerships in the future
Year(s) Of Engagement Activity 2017
URL https://content.biostratamarketing.com/blog/a-round-up-of-the-key-highlights-from-biobeat17
 
Description Talk at New Scientist Live 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Invited talk at New Scientists Live entitled 'The tiny universe inside your cells' with associated promotion of the Etch-a-Cell Citizen Science platform.
Year(s) Of Engagement Activity 2017
URL https://live.newscientist.com/talks/the-tiny-universe-inside-your-cells
 
Description Webinar interview 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Science Select Interview which talked about the need for higher resolution imaging and merging of technologies.
Year(s) Of Engagement Activity 2016
URL http://www.selectscience.net/editorialfeatures.aspx?editorialFeatureID=19&videoID=3304&utm_source=Ma...