Imaging cellular structure and function beyond the diffraction limit

Lead Research Organisation: Imperial College London
Department Name: Institute of Clinical Sciences

Abstract

Super-resolved microscopy (SRM) represents a revolution in optical imaging with profound implications for the life sciences. It allows researchers to visualise biological structure and function on almost the molecular scale in live cells and organisms. Typically biological processes are visualised by attaching fluorescent molecules to proteins of interest forming images from the light emitted by these fluorescent "labels". The optical resolution, which defines the smallest structures that can be visualised, has long been regarded as fundamentally limited to >200 nm by the diffraction of light waves. Recent breakthroughs in fluorescence microscopy, however, have exploited the ability to systematically switch the emission of fluorescent molecules on and off in order to break the "diffraction limit" and achieve resolutions down to 10's of nm - almost the scale of the typical biological proteins that are involved in signalling processes that determine the fate of cells and organisms and so are essential to disease processes.

This project builds on the partnership between the MRC Clinical Sciences Centre (CSC) and Imperial College London to develop a range of SRM instruments to advance our understanding of epigenetics, regulation of genes and cells, immunology and metabolism by resolving fundamental biological mechanisms inside the nucleus, inside live cells and at the synapses of interacting cells. It also aims to establish super-resolved microscopy as a widely accessible tool for the research community at the CSC and Imperial.

The first SRM technique would be structured illumination microscopy (SIM) that entails illuminating samples with patterns of light and observing the interference patterns between these patterns and the structure of the sample. This effectively doubles the resolution of a normal microscope. The second approach is an advance on laser scanning confocal microscopy, in which as sample is imaged by scanning a focused laser beam across a sample and collecting the fluorescence pixel by pixel through a pinhole that blocks out of focus or scattered light. The size of this focussed laser beam spot defines the resolution of this microscope. By using a second, collinear, specially shaped laser beam that is scanned synchronously and can switch off the fluorescence from molecules around the outside of the first beam focus, the fluorescence excitation is restricted to a smaller spot than the diffraction limit and thus resolutions below 50 nm can be realised. Because this technique can block scattered or out of focus light, it is particularly suitable for imaging in live organisms. The third SRM modality relies on having only a small fraction of the fluorescent labels being switched on at any time such that they can be treated like arrays of single molecules. Their individual positions can be determined by locating the centres of the "blobs" that represent each molecule in an image - which is possible to a precision of a few nm. By sequentially switching on different subsets of the fluorescent labels and determining their individual positions, one can build up a super-resolved "image" that is simply the map of the locations of all the fluorescent molecules.

In this proposal, we will develop and apply these new imaging techniques to visualise biological structures and processes that are simply too small or inaccessible for conventional microscopes. Starting at the surface of cells, researchers will be able to observe how immune cells communicate and nerve cells interact with each other, and how tumour cells relate to normal cells. Looking inside cells, we will visualise signals that communicate the energy status of cells and how the genome is organised. It will be possible to resolve how chromosomes behave in developing germ cells, which is relevant to the most frequent causes of infertility and birth defects. The research will deliver new insights into both normal biological processes and disease states.

Technical Summary

This partnership aims to combine strengths in basic science underlying human disease and imaging technology to develop capabilities in super-resolution microscopy (SRM) in order to study fundamental biological mechanisms inside cell nuclei, inside live cells and at the synapses of interacting cells.
Initially we would establish a commercial SIM microscope to provide rapid SRM of cells with existing labels. This would enable users to rapidly gain expertise in SRM, e.g. through studies of chromatin organisation and reprogramming of lymphocytes by ES cells. We would also implement rapid FLIM FRET, e.g. for directly correlation of SRM of changes in subcellular structure with cell signalling.
In parallel the Photonics team would extend their 3-D STED microscopy to live cells with GFP and YFP and also implement RESOLFT using, e.g. rsGFP. This will utilise novel fibre-laser based excitation/depletion/switching lasers developed in-house, as well as SLM technology for beam shaping and adaptive optical correction of aberrations. A key application would be to image synapses between live cells - noting that these can lie in arbitrary planes not readily accessible to other SRM techniques. Progressing to intravital imaging, we would also develop a STED/RESOLFT microscope for to study the formation and function of neurological synapses in live in mice.
We would also upgrade our in-house developed TIRF-based PALM/STORM systems to live cell imaging using emerging algorithms to separate signals from closely packed fluorophores and implement 3-D PALM/STORM using, e.g. axial localisation and oblique light sheet illumination. We would then replicate this system in the CSC for studies of nuclear reorganisation during meiosis in C. Elegans and for other subnuclear imaging applications.
The in-house development of SRM technology makes it cost effective and inherently upgradeable, enabling it to be sustained at the cutting edge for multiuser access beyond the project's end.

Planned Impact

This significant project would have impact derived from the new technology to be developed and from the novel biology to be addressed. It would have a major impact on the CSC and Imperial College through the establishment of SRM and its development to functionality beyond the state of the art. As the partnership develops, the benefits of this project will extend beyond the initial group of investigators, with "stable" SRM technologies becoming embedded as multiuser facilities within the well-established FILM in South Kensington and within the microscopy facility at the CSC on the Hammersmith site. We would also explore the possibility of becoming a node of the ESFRI Euro-BioImaging (EuBI) initiative, which would provide another avenue for broader dissemination of our technology. Thus this project would "raise consciousness" about SRM and the new opportunities it presents and highlight expertise in its application throughout Imperial and beyond.
The technology and software tools that we propose to develop and integrate could be applied by others for SRM and related microscopy techniques. They are also likely to find diverse applications in a range of optical instrumentation. With respect to technology development, the Photonics Group has a strong track record of interacting with industry particularly for fluorescence imaging techniques and for translating novel fibre based scientific laser source concepts into technologically attractive and commercially viable prototypes. A particular example of this are the high power all-fibre-based visible light sources providing pulsed radiation with variable repetition rate and picosecond pulse durations that can find application in a wide range of instrumentation besides SRM, including, e.g. wide-field and laser scanning confocal microscopes, FLIM systems, time-resolved fluorometers, flow cytometers and displays. The SLM techniques for aberration correction could also find application in diverse optical instruments including for imaging and metrology and the software tools to be developed could be applied in, or adapted to, other contexts. We would make software tools publically available, probably via the OME platform, once they are robust. The programme is also likely to deliver a range of novel labels and probes for SRM in different systems. As we publish and disseminate our work, these probes will be made available to the scientific community and will therefore become a resource for future research.
The biological components of the proposal cover a range of areas that are important for health and disease, including immune and neurological synapse formation and function, cancer biology, lymphocyte reprogramming and chromatin reorganisation. While our research is primarily aimed at making fundamental insights into the biology of these systems, both in normal cells/ tissues and following genetic or pharmacological manipulation, the use of SRM would provide a level of detail for structures such as neurological and immune synapses that has never before been possible and this may have longer term implications for the understanding of disease mechanisms and the development of new therapeutics. Combining functional insights from FRET and FLIM with structural insights from SRM for the first time could bridge the resolution gap between biochemistry and imaging, potentially providing a step change in our understanding of structure/ function relationships in these important systems and helping us better understand both normal and disease states. Thus the immediate impact would be to the academic research community but in the longer term the project could stimulate the development of new therapies.
The knowledge and skills gained by the staff working on this project would directly benefit them in terms of increasing their potential for research impact and future employment and could benefit the biophotonics instrumentation and biomedical research communities who could employ them.

Publications

10 25 50
 
Description BBSRC Impact Acceleration Account: Open, modular, accessible, super-resolved microscopy
Amount £47,212 (GBP)
Funding ID BB/S506667/1. 
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 06/2018 
End 11/2019
 
Description Imperial Confidence in Concept (ICiC) Scheme
Amount £75,000 (GBP)
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 08/2019 
End 12/2020
 
Description Research England GCRF fund: openScope - phase 1
Amount £174,089 (GBP)
Organisation Imperial College London 
Sector Academic/University
Country United Kingdom
Start 11/2018 
End 09/2019
 
Description Research England devolved Global Challenges Research funding
Amount £132,933 (GBP)
Organisation United Kingdom Research and Innovation 
Department Global Challenges Research Fund
Sector Public
Country United Kingdom
Start 08/2019 
End 07/2020
 
Title New applications of super resolved microscopy 
Description We are using super resolution microscopy to investigate how chromosomes become structurally organized around proteinaceous axial elements at the onset of meiosis, and how these structures are remodeled at later meiotic stages. We are particularly interested in structural changes that occur in the context of fully paired homologues, when homologous axial elements are separated by a distance of about 100 nm, and therefore can not be resolved by conventional microscopy. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2015 
Provided To Others? Yes  
Impact Angel Luis Jaso-Tamame, a second year PhD student, has successfully developed methods to visualize C. elegans meiotic chromosomes using the Zeiss ELYRA SIM and the Leica TCS SP8 STED 3X systems at the CSC. Using different approaches to label meiotic proteins (tagging with fluorochromes by CRISPR or single-copy transgenes, and protein visualization with primary antibodies), we have been able to resolve individual axial elements from three-dimensionally intact germ lines with the SIM and STED systems. For example, we have used the SIM system to investigate how WAPL, a protein that regulates the association of cohesin with chromosomes, affects the removal of different cohesin complexes during meiosis. We continue to use the SIM and STED systems to investigate how different protein complexes modulate the structure of meiotic chromosomes, and we are also aiming to develop protocols to implement PALM and STORM, which should provide better resolution that SIM or STED, to visualize meiotic chromosomes. People impacted by training in the use of SRM microscopes are Angel Luis Jaso Tamame - Meiosis Consuelo Barroso - Meiosis Lucien West - Neuroplasticity and Disease Katerina Papadopoulou - Neuroplasticity and Disease Rahuman Sheriff - Quantitative Cell Biology Buhe Nashun - Petra Hajkova Group Martin Billman - Single Molecule Imaging Nicholas Darvill - Single Molecule Imaging Rosa Maria Porreca - Telomere Replication and Stability Xi-Ming Sun - Quantitative Gene Expression Anne Celine Kohler - Lymphocyte Development Group Alex Savell - Paul French Group Adam Cawte - Single Molecule Imaging Frederik Goerlitz - Paul French Group The refurbished facility before installation of the microscopes: https://imperiallondon-my.sharepoint.com/personal/alisini_ic_ac_uk/Documents/one%20pic.jpg https://imperiallondon-my.sharepoint.com/personal/alisini_ic_ac_uk/Documents/two%20pic.png 
URL https://imperiallondon-my.sharepoint.com/personal/alisini_ic_ac_uk/Documents/one%20pic.jpg
 
Description Cairn Research Ltd 
Organisation CAIRN Research Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have developed TIRF and STORM microscopy techniques which we have published. Cairn have implemented these ideas into their product development and will be able to provide the benefit of low-cost TIRF and STORM microscopy to the community. We have developed optical projection tomography (OPT) technology which we have published. Cairn are intending to introduce our ideas into their product development and will be able to provide the benefit of low-cost OPT to the community.
Collaborator Contribution Cairn have advised on components and practical implementations of various aspects of our instruments and have provided us with prototypes at significant discounts that have been customised to our requirements. We have advised them on key research issues for microscopy and designed open source optical instrumentation for which they are now making components available on a commercial basis to the wider user community who wish to access the technology but cannot or do not wish to fabricate it themselves. This instrumentation is intended to work with open source software, including our open source software. Cairn will now provide our openFrame microscope that can be used to implement low cost STORM and TIRF microscopy and almost any other microscope modality. openFrame is our original concept and the design has been refined with input from Cairn. Cairn will also provide low-cost OPT instrumentation based significantly on our design.
Impact The instrumentation development is essentially optical physics but the applications are multidisciplinary - mainly biomedical
Start Year 2015
 
Description Imaging optical chemical sensors using FRET-FLIM in vivo 
Organisation Imperial College London
Department Department of Bioengineering
Country United Kingdom 
Sector Academic/University 
PI Contribution we proposed the project
Collaborator Contribution they will provide key expertise
Impact A PhD student has been recruited
Start Year 2012
 
Description Imaging optical chemical sensors using FRET-FLIM in vivo 
Organisation Imperial College London
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution we proposed the project
Collaborator Contribution they will provide key expertise
Impact A PhD student has been recruited
Start Year 2012
 
Description Imaging optical chemical sensors using FRET-FLIM in vivo 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution we proposed the project
Collaborator Contribution they will provide key expertise
Impact A PhD student has been recruited
Start Year 2012
 
Description Imperial College - MRC 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Enhancing collaboration between Imperial College London and the MRC Clinical Sciences Centre.
Collaborator Contribution Collaboration.
Impact Enhanced collaboration and publication.
Start Year 2013
 
Description Imperial College - MRC 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution Enhancing collaboration between Imperial College London and the MRC Clinical Sciences Centre.
Collaborator Contribution Collaboration.
Impact Enhanced collaboration and publication.
Start Year 2013
 
Title USE OF PI3K M-TOR AND AKT INHIBITORS TO INDUCE FOXP3 EXPRESSION AND GENERATE REGULATORY T CELLS 
Description The invention relates to a method of inducing Foxp3 expression in a T cell comprising (i) stimulating a T cell (ii) inhibiting signalling via PI3K alpha or PI3K delta or m-TOR or Akt in said T cell, wherein said inhibition is commenced 10 to 22 hours after the stimulation of (i). The invention also relates to certain uses of PI3K inhibitors, PI3K inhibitors for particular uses, and kits. 
IP Reference US9314485 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact The present invention seeks to overcome problem(s) associated with the prior art. It is to be noted that it is an advantage of the present invention that de novo Foxp3 expression is being induced. This is contrasted with the situation in the prior art where the best effect shown to date has been the expansion of T cells which are already expressing Foxp3. The opportunity to actively induce Foxp3 expression in cells which are not currently expressing it is a significant advantage of the present invention. We show that small molecule inhibitors of the PI3K/mTOR pathway rapidly and efficiently induce the de novo expression of Foxp3, the signature transcription factor of the Treg cell lineage. This enables new therapeutic approaches to be applied to clinical settings where the de novo generation of Treg cells is desirable, such as the prevention and the treatment of autoimmune diseases. Many of these diseases are of major clinical significance, such as rheumatoid arthritis and type I diabetes. Thus the industrial application and utility of the invention flow from these significant healthcare applications.
 
Title Accelerated single molecule localisation using HPC 
Description We have implemented parallelised single molecule localisation for the first time to our knowledge on a high performance cluster (HPC) . We developed scripts to partition large single molecule localisation microscopy (SMLM) data sets and process them in parallel using ThunderSTORM in order to decrease the total data processing time. This can be scaled to a large number of HPC nodes and therefore provide almost arbitrary acceleration, which will be important for high throughput SMLM applications. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2017 
Impact As well as implenting this at Imperial College london, we have also now implemented it at the Francis Crick Instutite where it makes possibl enew high throuhgput applications of SMLM. We are developing an high throughput application of easySTORM - our robust and low-cost super-resolved microscopy technique. 
URL https://github.com/ImperialCollegeLondon/HPC_STORM
 
Title SIM+FLIM microscope platform 
Description We have developed a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact We have applied this SIM+FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle. We have also used it to study interactions between organelles where FLIM/FRET can confirm proximity of nanostructures that is not resolvable by SIM. 
 
Title easySLM-STED 
Description We have developed a novel set-up for STED microscopy that provides a straightforward alignment procedure that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams to provide phase modulation of the beam profiles and correction of optical aberrations. We have shown that this approach can be used to extend the field of view for STED microscopy by correcting chromatic aberration that otherwise leads to walk-off between the focused excitation and depletion beams. We have extended this arrangement to increase the imaging speed through multibeam excitation and depletion. Fine adjustments to the alignment can be accomplished using the SLM only, conferring the potential for automation. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2018 
Impact We have applied easySLM-STED to image meiotic chromosomes in from C.elegans nematodes expressing the axial element component HTP1 labelled with GFP. The easySLM-STED microscope was able to resolve the alignment of homologous axial elements containing HTP-1 at a distance of about 100nm. 
 
Title easySTORM upgrade for commercial microscopes 
Description We have developed a low-cost modular upgrade of a commecial fluoresence microscope to easySTORM for super-resolved imaging 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2019 
Impact This upgrade has been installed at the Indian institute for technology in Guwahati and at the Shenzhen Technology University 
 
Title openFrame microscope frame 
Description openFrame is a microscope frame that is part of our low-cost modular microscopy platform oringally designed to work with easySTORM but extendable to almost any microscopy modality 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2020 
Impact Cairn Research Ltd have strted manufacturing this concept to use it as a platform for their own micrscope technology 
URL https://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/fluorescence-micros...
 
Description Biophotonics Summer School Hven 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I lectured on the Biophotonics Summer School organised in Hven, Sweden every two years. This is probably the highest level school int he field in terms of the lecturers and attracts students from all over the world. I presented the basic principles of fluorescence microscopy and our latest research in multidimensional fluorescence imaging including super-resolved microscopy, FLIM and optical tomography as well as clinical applications
Year(s) Of Engagement Activity 2015
URL http://www.biop.dk/biophotonics15/school/school.asp?page=main_school_lecturers
 
Description MDFI user-orientated workshop 2017 
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 This one day user-orientated workshop entitled "Multidimensional fluorescence imaging technology: super-resolution, HCA and preclinical imaging" showcased the biophotonics instrumentation and applications of the technologies developed in the Photonics Group at Imperial College London. It attracted 80 participants including 11 from industry and 40 for other research organisation. The talks and posters were well received and this event led to further collaborations, including with industry.
Year(s) Of Engagement Activity 2017
 
Description MDFIM user-orientated workshop 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Multidimensional fluorescence imaging and metrology user-orientated workshop held at Physics Department, Imperial College London on 27/09/13.
~50 external visitors attended talks, posters and networking sessions. The aims were to identify new users of our research, to reinforce relationships with existing users, to identify new research partners, sponsors and opportunities. The event was also a showcase for our research progress and students and staff and a source of feedback to inform our ongoing technology development.

Three companies expressed interest in commercialising aspects of our work
Several external research partners learned about our capabilities and suggest new research collaborations
We received excellent feedback about quality of work presented and of presentations
Year(s) Of Engagement Activity 2013
URL http://www3.imperial.ac.uk/photonics/events/photworkshop
 
Description SRM workshop at Hammersmith Hospital 01/03/17 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact We organised a workshop to show case the new super-resolved microscopy and FLIM/FRET microscopy capabilities that we have developed during this project. We presented the technical advances and the capabilities that our life scientists could use for their research. We also arranged for our life scientist colleagues to explain how they have been using the technology and how they would like it to further develop.
Year(s) Of Engagement Activity 2017
 
Description School visit (Horsted Keynes) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I presented the impact of biophotonics and imaging technology on the world around us and on medicine and drug discovery
Year(s) Of Engagement Activity 2017
 
Description Superresolution Microscopy Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Study participants or study members
Results and Impact A workshop was organised, involving professionals from the CSC, the Imperial College in London, and open to students.
The meeting was productive in terms of interactions within the institutes and to spread interest in the project outcomes among colleagues.
Year(s) Of Engagement Activity 2015