A super-resolution multi-scale in vitro and in vivo imaging platform at Harwell: building models of development and disease from molecules to mammals

Lead Research Organisation: Research Complex at Harwell

Abstract

Capturing the 'why' of a disease and the 'how' of a treatment requires building a model of the disease and its evolution during treatment. This model ideally needs to capture everything, from the interplay of a single molecule with a molecular network, to inter-cellular communication networks that underpin the development of a phenotype. To reach this goal we need to bridge the different scales on which biology operates - the molecular, the cellular and the whole organism. One way to do this is to image cells and organisms at different scales: (i) the nanoscale to show how cells are organised at the molecular level and how molecular therapies affect molecular interactions; (ii) the microscale to show the functions of cellular organelles and their reorganisation under therapeutic challenge; (iii) the mesoscale to investigate cellular behaviour during development and/or the progress of disease; (iv) and the whole organisms level, to monitor changes and the well-being of the entire organism in response to therapy (e.g. tumour remission). Only when these jigsaw pieces are put together should one be able to understand how mutated genes and proteins affect development, predict how the "next generation" therapies can deliver breakthrough advances and elucidate how therapeutic agents can be delivered to focal areas of disease to maximize clinical benefit while limiting side effects.
Cells were discovered by Robert Hooke (1635-1703) under the optical microscope. Lord Rayleigh (1842-1919) empirically determined that the resolution of a diffraction-limited microscope can be no better than ~ 1/2 of the wavelength of light (i.e. >200 nm), which defines the microscale. For centuries this was a fundamental limitation of light microscopy, as this resolution is insufficient to resolve the nanoscale processes underpinning biology. Despite this, through the availability of many organic labels and the discovery of green fluorescent protein, fluorescence microscopy has been fundamental for decades to many of the in vitro-based key discoveries in the biomedical sciences.
The 'resolution limit' of light microscopy was broken at the end of the last millennium using a challenging technique, stimulated emission depletion microscopy, which showed ~20 nm resolution, followed by the less damaging structured illumination microscopy with 90 nm resolution. During the last decade, 'simpler' modes of super-resolution microscopy achieved similar resolutions by using the fundamental principle that molecules are much smaller than the wavelength of light and therefore can be considered 'single point' emitters. This is very important because their position in space can therefore be determined with nanometre accuracy via deconvolution of the 'blob'-like spot image created by the microscope optics, which incidentally is the origin of the poor resolution associated with light microscopy. High resolution images at the nanoscale are formed by putting together individual molecular images, a time-consuming process which nevertheless has already delivered spectacular results.
High resolution imaging at the mesoscale is critical to understand basic mammalian biology. OCT was developed to address the need for fast imaging tools to characterise the inter-play between cells in a whole organisms in order to model human pathophysiology, and assess benefits resulting from therapeutic treatments in pre-clinical research.
We have formed an interdisciplinary partnership that seeks to exploit a new generation of world-leading super-resolution microscopy in combination with state-of-the-art in vivo imaging methods (like OCT). Our principle is to break the barriers between fields to ease the exploitation of these new technologies by the wider biomedical community and to place the UK at the imaging forefront. The interdisciplinary environment and concentration of scientists at the Harwell Campus will help in our efforts to underpin fundamental discoveries in the next decade.

Technical Summary

We have identified three developments to our current facilities to make the proposed multi-scale in vitro-in vivo platform world-leading:

Firstly, to purchase a top-of-the-range 3D super-resolution microscope that combines structured illumination microscopy (SIM) with fluorescence localisation microscopies such as stochastic optical reconstruction microscopy (STORM) for live cell imaging to satisfy the strong demand for this technology at the earliest opportunity. Secondly, to establish a 4D ultrafast OCT system to provide much needed access to high-throughput in vivo imaging of embryonic and adult organ systems. Not only there is an urgent need in our communities to have access to these systems but their availability is also essential to make the proposed multi-scale platform internationally competitive.

Our choice of commercial super-resolution system has been directed by the ideal of imaging live cells in real-time. However, all the microscopes available have a number of technical limitations that require making one or more of the following compromises: between spatial and temporal resolution, between speed, resolution and imaged sample volume and between photodamage and resolution. The third development we propose is to build the first truly 3D super-resolution microscope that will obtain images with a resolution of <20 nm both axially and laterally and that will collect data from a 3D volume of cell dimensions in a single shot rather than imaging planes within the sample individually. This microscope will be able to image multiple targets within the entire cell volume at this resolution in all three spatial axes and subsecond temporal resolution. At this time scale a wealth of new science will become accessible as we will be able to follow with nanoscale resolution in real-time a multitude of cellular process and molecular interactions as they occur. This will make the proposed imaging platform internationally leading.

Publications

10 25 50
 
Description An automated macromolecular structure-based approach to personalised anti-cancer therapy
Amount £576,054 (GBP)
Funding ID ST/S000682/1 
Organisation Science and Technologies Facilities Council (STFC) 
Sector Academic/University
Country United Kingdom
Start 09/2018 
End 09/2021
 
Description Bearing the context in mind: A cryo FIB-SEM based CLEM workflow to investigate relationships between molecular interactions and ultrastructure
Amount £544,802 (GBP)
Funding ID BB/S019553/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 08/2020
 
Title Development of OCT Platform at Harwell 
Description From the point of view of refining mouse-based research, the development of the OCT platform at the MRC Harwell Institute has already demonstrated a great potential in the improved capability of detecting small abnormalities in the eye morphology. 
Type Of Material Improvements to research infrastructure 
Year Produced 2016 
Provided To Others? Yes  
Impact The quality of the data generated by our OCT procedure, coupled with the Mary Lyon Centre's high standards in animal phenotyping, will allow on site researchers and external scientists to conduct their studies on eye morphology diseases mouse models at the highest levels. 
 
Title Development of cryo-super resolution optical microscopy 
Description Using special optics we have developed high numerical aperture fluorescence microscopy under cryogenic conditions. This is crucial for super-resolution microscopy at low temperature, and will be an important enabler for correlative light and electron microscopy with much improved optical resolution. A publication is in the final stages of preparation. 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact The technique is available to researchers as part of the portfolio of methods offered by the CLF Octopus facility in RCaH. There has been significant interest and a number of proposals to use the technique have already been assessed. 
 
Description Collaboration on correlative microscopy development 
Organisation Diamond Light Source
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Howard Hughes Medical Institute
Department Janelia Research Campus
Country United States 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Rosalind Franklin Institute
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration on correlative microscopy development 
Organisation Yale University
Country United States 
Sector Academic/University 
PI Contribution Development of advanced optical microscopy methods including super-resolution imaging under cryogenic conditions.
Collaborator Contribution Expertise in electron microscopy, x-ray microscopy, and 3D super-resolution microscopy.
Impact A cryo super-resolution microscope is now operating for users in the Central Laser Facility (Research Complex at Harwell). This is a multi-disciplinary partnership combining physics (microscopy), engineering (sample stages etc.), biology (sample preparation), and chemistry (labelling).
Start Year 2017
 
Description Collaboration with AstraZeneca on EGFR-drug interactions 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Provision of expertise in FLImP technique and its application to the study of EGFR.
Collaborator Contribution Expertise in drug development and potentially access to drug molecules for proposed CLASP grant programme (see below).
Impact AZ are setting up a single molecule system in house. They are partners on an STFC CLASP proposal that aims to exploit single molecule techniques (FLImP) for drug discovery and development (outline proposal accepted, awaiting decision on grant).
Start Year 2017
 
Description Collaboration with Oxford NanoImaging on Single Molecule techniques 
Organisation Oxford Nanoimaging
PI Contribution Provision of expertise in single molecule techniques (FLImP).
Collaborator Contribution Expertise in development of compact microscope systems.
Impact Joint proposal to STFC CLASP scheme for funding to develop single molecule techniques for targeted cancer therapies. Outline proposal accepted. Awaiting decision on award.
Start Year 2017
 
Title Super-resolution microscopy 
Description This patent describes the use of solid immersion lenses for super-resolution microscopy at cryogenic temperatures. It solves a long-standing problem of how to achieve the necessary high numerical aperture optics without the use of immersion oil. It is simple and cheap and should find a wide range of applications. 
IP Reference 1704275.5 
Protection Patent application published
Year Protection Granted 2017
Licensed No
Impact The main impact to date is the availability of cryo-super resolution microscopy on the CLF Octopus facility in the Research Complex at Harwell. This new technique is available to UK adademics through an open peer review access procedure.
 
Title FLImP single molecule analysis software 
Description The FLImP software analyses single molecule photobleaching tracks and determines inter- and intra-molecular separations with ~ 5 nm resolution. 
Type Of Technology Software 
Year Produced 2015 
Impact A number of publications using this technique, including two in Nature Communications. 
 
Description Correlative Imaging Workshop 
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 A workshop was held in the Research Compex at Harwell and Diamond Light Source, consisting of a day of talks from international speakers and two days of "hands-on" experience in use of light, electron, and x-ray microscopy for postgraduate students and early career researchers.
Year(s) Of Engagement Activity 2017
 
Description Harwell Campus Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Contribution to Harwell Campus open days, in which the laboratory was visited by 16000 members of the public. Special events were also held for schools and VIPs.

We set up and manned displays explaining our research.
Year(s) Of Engagement Activity 2015
URL http://harwellcampus.com/open-days/
 
Description Multiple school visits to Rutherford APpleton Laboratory and the Research Complex at Harwell 
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 Discussion with visitors about my research area and optical imaging in general.

Ongoing interest from schools in sending further pupils to the lab.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description Public engagement in OCT imaging 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Throughout the lifetime of the project, we have attended a number of meetings and events publicising our activity with the OCT techniques, providing information on the accessibility of the OCT and on all the typical OCT examinations available. We have attended the annual Bio-Imaging Festival in Oxford, presenting posters highlighting the importance of the OCT technique on eye phenotypes discovery.
Year(s) Of Engagement Activity 2015,2016