Super-Beacons and Beacon-STORM: a new generation of small tunable photoswitching probes and Super-Resolution approaches.

Lead Research Organisation: University College London
Department Name: Lab for Molecular Cell Bio MRC-UCL

Abstract

Microscopy has been the major tool in cell biology. Its inception in the 16th century led to the first 'wave of discovery' - the finding and comprehension of cells and their internal structure. However, fundamental limitations on modern light microscopes (e.g. widefield and confocal) prevent us from accurately resolving structures smaller than 300 nm. It took three centuries to achieve a second 'wave of discovery' - the development of electron microscopes (EM) able to bypass this resolution limit, offering a new view into the realm of small biological complexes, e.g. endocytic vesicles and viruses. Nevertheless there are two main limitations to EM as it does not allow to: 1) image live-cells and 2) use molecules labelled with fluorescent tags. We are now at the forefront of a 'third wave of discovery' brought about by the development of Super-Resolution light microscopy - a range of methods that approach the resolution accuracy of EM but with the capability of live cell imaging and molecule-specific labelling. However, Super-Resolution imaging is not trivial and can only be achieved by a fine balance between three key components: 1) highly-sensitive often bespoke microscopes; 2) optimised fluorescent labels; 3) advanced computational analysis. So far, the development of these three factors by the research community has not been fully coupled - e.g. we have reached a stage where computer processes and hardware have been formalised for video-rate high-speed Super-Resolution, but there is still a lack of suitable non-toxic fluorescent probes. This hinders the potential of Super-Resolution microscopy as a widespread live-cell imaging tool.

This project addresses this issue, by integrating the development of 1) a new generation of small probes with tuneable photoswtiching kinetics designed for high-speed low-toxicity Super-Resolution imaging; 2) a high-speed imaging system able to modify the imaging microenvironment by adjusting probe properties in real-time; 3) Super-Resolution acquisition software able to make data-driven decisions to optimally balance the probe's photokinetics for best speed and resolution.

Recently, we have prototyped a new type of probe called Super-Beacon. Its structural properties allow to convert almost any synthetic fluorophore into high-performance probes with adjustable photokinetics. Based on the principles of Super-Beacons, we aim to design a new generation of probes optimised for high-speed multi-colour Super-Resolution microscopy. In parallel, we will develop a new analytical (software) and imaging (optical hardware) framework - called Beacon-STORM (BeaST) - that takes advantage of Super-Beacons to achieve an improved level of resolution, speed and low photo- and chemical-damage in Super-Resolution microscopy. Keeping up with our track record of providing critical tools enabling Super-Resolution imaging to the research community, we will follow an open access policy and provide the tools and framework for researchers to easily adapt and use Super-Beacons and BeaST for their own research.

As a proof-of-principle of the application of these two highly complementary technologies, we will target a fundamental and open question in eukaryotic cell biology - what is the trigger and required structural remodelling of receptors at the cell membrane to promote clathrin-mediated endocytosis? Using viral like particles as model cargo, we will address this question by super-resolving in vivo the nanoarchitecture of early endocytic sites, mapping the cellular factors involved in vesicular formation. This question can only be optimally answered by an approach such as the one proposed, as it requires a system capable of resolving, in live-cells, the vesicle formation site nano-organization with minimal disruption of its behaviour. Understanding this interplay is critical to uncover the basis of endocytosis and understand how cells deal with signalling noise, such as stochastic receptor clustering.

Technical Summary

We aim to improve Super-Resolution Localisation Microscopy, by developing a novel, dynamically tunable imaging framework for live- and fixed-cell high-speed multi-colour Super-Resolution. The project entails the development and validation of a novel type of modular Super-Resolution probe of simple synthesis, allowing us to easily 'plug-in' most available synthetic fluorescent dyes into a linker scaffold that induces photoswitching by transient high-efficiency quenching. This feature avoids the need for non-physiologic (generally toxic) photoswitching inducing buffers, thus improving live-cell Super-Resolution imaging. A hardware-software imaging framework will be developed to take advantage of the specific photoswitching properties of these probes. We aim to fix a major imbalance between the properties of current probes for Super-Resolution Localisation Microscopy (bulky and nearly uncontrollable photoswitching dynamics) and imaging apparatus whose performance is constrained by suboptimal probe photokinetics and size. These developments will enable a step change in the use of Super-Resolution Localisation Microscopy across multiple fields including cell biology, virology, biophysics and biochemistry.
The project will be developed in the LMCB at UCL, allowing these developments to be directly exploited and validated by researchers in cell biology, also strategically benefiting from the local interdisciplinary community. The project will support the formation of Ricardo Henriques' group (biophysics, optical physics) as a young investigator and will nucleate collaborations with partners such as Dr. S. Cox (biophysics, KCL) and Dr. M. Marsh group (cell biology and virology, UCL). Jointly, we will apply a cell biology and physics view to unravel the mechanisms behind clathrin-mediated endocytosis. This final goal will serve as an exemplary demonstration of the use of Super-Beacons and BeaST, accelerating their application in the larger biotechnology sector.

Planned Impact

Academic: We aim to deliver to researchers a novel imaging approach that can be easily implemented in existing imaging systems and considerably enhances Super-Resolution localisation microscopy. These will allow experiments in live-cell imaging to explore the nanoarchitecture of cells at scales (~1-30nm) inaccessible to conventional light imaging approaches.

Training: The PDRA to be recruited will receive continual cross-disciplinary training and mentoring, which will aid his/her progression towards an independent group leader position. In addition, the PDRA will benefit from generic skills gained on training courses at UCL, through co- authoring papers, grants and reviews and by presenting this work at international meetings. RH is actively involved in interdisciplinary training activities at UCL. RH is the academic lead for imaging in BioSciences, contributing to the BBSRC LIDo, UCL CoMPLEX and MRC LMCB MRes-PhD training programme. The project described here will be ideal for introducing students from different backgrounds to interdisciplinary research in the life and physical sciences. Moreover, a large number of students will benefit from involvement in this interdisciplinary level research through rotation projects, MRes and tutorial activities associated with these programmes. Similarly, undergraduates will be exposed to this work through internships and short projects. Finally, by publicising this work, we expect to attract better and brighter students and researchers to the UK.

Commercial: It will be possible to rapidly translate the probes, analytics and hardware developed from this project into imaging applications of interest to biotechnology, microscopy and imaging companies. In addition, the novel BeaST method can be developed to achieve high-speed drug screening of cell or tissue features only visible by Super-Resolution microscopy, which is applicable within pharma and small biotech industry. These new methods will open the door for new diagnostic approaches requiring Super-Resolution (e.g. molecular interactions impairment). We also envision the application of the Super-Beacons probes as customizable nano-environment sensors as their photoswitching kinetics can be made to report changes in the surrounding thermal and chemical settings.

General Societal and Economical Impact: Microscopy approaches and instrumentation is one of the fastest growing areas in biomedical research, critical not only for cell biology, but for the direct discovery of new molecular targets mitigating human disease. Over the past decades, the UK has been a world leader in these developments. This project will enable and supply new approaches crucial for enhancing microscopy, supplying UK research with novel state-of-the-art and experimental methods - a key attractor for industrial R&D collaborations - and empowering research in the UK with some of the most advanced imaging facilities in Europe. Locally, UCL (where this project will be housed) has made a commitment to make the interface between medicine, biophysics and imaging a key priority, contributing to the long-term sustainability of these studies and serving as a seeding source for collaborations with our partners such as the new Francis Crick Institute and NHS Hospitals (e.g. Royal Free Hospital).

Outreach: RH has been involved in interactions with the wider community through media appearances, public discussions and school visits. Through this type of outreach we expect this work to reach a wide audience; giving the public a better understanding of how next-generation imaging technology may impact health and disease. Moreover, through our involvement in EMBO, EU and UK-South Africa networks, we expect this work to reach the global scientific community.

Publications

10 25 50
 
Description This grant entails the development of novel probes and analytical software to enable live-cell super-resolution microscopy. Until now live-cell super-resolution imaging studies have remain hampered by the requirement for intense illumination that is usually phototoxic. Both the new probes being prototyped and new algorithm address this problem, allowing the study of normal cellular behaviour uncorrupted by high-intensity sample illumination. In this aspect, we already published the Super-Resolution Radial Fluctuations approach, a high-impact paper addressing the analytical side of this problem. In parallel we are already achieving beneficial results with the new class of fluorescent probes being developed.

Support from the BBSRC IAA allowed the research group led by Dr Henriques from UCL's MRC Laboratory for Molecular Cell Biology to strengthen a translational partnership with a biotechnology company - Intelligent Imaging Innovations (3i), leading to a research and development partnership.
Exploitation Route All advancements planned in this grant, both in therms of new analytics and biochemistry, will be made available openly to researchers.
Sectors Chemicals,Digital/Communication/Information Technologies (including Software),Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.ucl.ac.uk/lmcb/users/ricardo-henriques
 
Description Impact Acceleration Account
Amount £10,000 (GBP)
Funding ID BB/IAA/UCL/15 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 07/2016
 
Description Andor Technology 
Organisation Andor Technology
Country United Kingdom 
Sector Private 
PI Contribution Developed the Super-Resolution Radial Fluctuations method which was used by Andor Technology to establish the worlds first Super-Resolution Cameras for fluorescence microscopy, which are now commercially available.
Collaborator Contribution Andor Technology has contributed with known-how and research financial support.
Impact This R&D collaboration has led to the development of a new generation of cameras for microscopy and spinning disk microscopes, these are now available commercially. This collaboration entails expertise in optical physics, cell biology and electrical engineering.
Start Year 2017
 
Description Andor Technology 
Organisation Andor Technology
Country United Kingdom 
Sector Private 
PI Contribution R&D partnership to implement analytical technologies developed into commercial turnkey systems.
Collaborator Contribution R&D partnership to implement analytical technologies developed into commercial turnkey systems.
Impact This collaboration has led to the translation of UCL developed technology into turn-key commercial hardware for biomedical research. As part of this collaboration Andor is bringing experimental kit and software to UCL that local researchers in biology and physics can use in their research.
Start Year 2017
 
Description Dr Darren Tomlinson 
Organisation University of Leeds
Department School of Chemistry Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution Validation of the small binders against CD4 receptors.
Collaborator Contribution Phage-display screening of small binders agains CD4 receptors.
Impact This collaboration is multi-disciplinary, involving the expertise of the Henriques lab (cell biology biophysics, mathematics and synthetic photobiochemistry) and the Tomlinson lab (biochemistry and cell biology).
Start Year 2015
 
Description Dr Mark Marsh 
Organisation University College London
Department MRC Laboratory for Molecular Cell Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Exchange of analytical resources and custom made algorithms. Data analysis.
Collaborator Contribution Exchange of cell lines, plasmids and other cell biology reagents.
Impact This collaboration is multi-disciplinary, involving the expertise of the Henriques lab (biophysics, mathematics and synthetic photobiochemistry) and the Marsh lab (virology and cell biology). We expect to have a joint publication submitted in 2016.
Start Year 2013
 
Description Dylan Owen - super-resolution studies on T-cell signalling 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on T-cell signalling.
Collaborator Contribution Joint development of the new super-resolution method SRRF and cell signalling studies.
Impact Joint publication: Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations N Gustafsson, S Culley, G Ashdown, DM Owen, PM Pereira, R Henriques Nature Communications 7
Start Year 2014
 
Description Eva-Maria Frickel - super-resolution studies on host-pathogen interactions 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on host-pathogen interactions.
Collaborator Contribution Studies on Toxoplasma gondii host-pathogen interactions.
Impact Joint publication: K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFN?-Stimulated Human Cells B Clough, JD Wright, PM Pereira, EM Hirst, AC Johnston, R Henriques, ... PLoS Pathogens 12 (11), e1006027
Start Year 2016
 
Description Helena Soares, CEDOC, Portugal - super-resolution studies on T-cell signalling 
Organisation New University of Lisbon
Department Chronic Diseases Research Centre (CEDOC)
Country Portugal 
Sector Academic/University 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on T-cell signalling.
Collaborator Contribution Joint studies on cell signalling.
Impact Joint publication: HIV-1 Nef Impairs the Formation of Calcium Membrane Territories Controlling the Signaling Nanoarchitecture at the Immunological Synapse JG Silva, NP Martins, R Henriques, H Soares The Journal of Immunology 197 (10), 4042-4052
Start Year 2013
 
Description Intelligent Imaging Innovations 
Organisation Intelligent Imaging Innovations Ltd
Country United Kingdom 
Sector Private 
PI Contribution R&D partnership to implement analytical technologies developed into commercial turnkey systems.
Collaborator Contribution R&D partnership to implement analytical technologies developed into commercial turnkey systems.
Impact This collaboration has led to the establishment of UCL as the R&D reference site for 3i in the UK. As part of this collaboration 3i is bringing experimental kit and software to UCL that local researchers in biology and physics can use in their research.
Start Year 2016
 
Description Musa Mhlanga, Univ Cape Town, South Africa - super-resolution studies on NF-kappaB signalling 
Organisation Council of Scientific and Industrial Research (CSIR)
Country South Africa 
Sector Academic/University 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on NF-kappaB signalling.
Collaborator Contribution Joint studies on cell signalling.
Impact Joint publication: Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti J Scholefield, R Henriques, AF Savulescu, E Fontan, A Boucharlat, ... Nature Communications 7
Start Year 2011
 
Description Musa Mhlanga, Univ Cape Town, South Africa - super-resolution studies on NF-kappaB signalling 
Organisation Pasteur Institute, Paris
Department Chemogenomic and Biological Screening
Country France 
Sector Charity/Non Profit 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on NF-kappaB signalling.
Collaborator Contribution Joint studies on cell signalling.
Impact Joint publication: Super-resolution microscopy reveals a preformed NEMO lattice structure that is collapsed in incontinentia pigmenti J Scholefield, R Henriques, AF Savulescu, E Fontan, A Boucharlat, ... Nature Communications 7
Start Year 2011
 
Description Serge Mostowy - super-resolution studies on host-pathogen interactions 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Development of optical and analytical technologies to enable quantitative super-resolution studies on host-pathogen interactions.
Collaborator Contribution Studies on Shigella sonnei host-pathogen interactions.
Impact Joint publication: Mitochondria mediate septin cage assembly to promote autophagy of Shigella A Sirianni, S Krokowski, D Lobato-Márquez, S Buranyi, J Pfanzelter, ... EMBO reports, e201541832
Start Year 2016
 
Title NanoJ 
Description In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was developed for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image reconstruction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact NanoJ provides the technological basis to a series of high-performance analytical algorithm for super-resolution microscopy data analysis 
URL https://iopscience.iop.org/article/10.1088/1361-6463/ab0261/meta
 
Title NanoJ-Fluidics 
Description Fluorescence microscopy can reveal all aspects of cellular mechanisms, from molecular details to dynamics, thanks to approaches such as super-resolution and live-cell imaging. Each of its modalities requires specific sample preparation and imaging conditions to obtain high-quality, artefact-free images, ultimately providing complementary information. Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows involving multiple sample preparation steps. We present a robust fluidics approach to automate complex sequences of treatment, labelling and imaging of live and fixed cells. Our open-source NanoJ-Fluidics system is based on low-cost LEGO hardware controlled by ImageJ-based software and can be directly adapted to any microscope, providing easy-to-implement high-content, multimodal imaging with high reproducibility. 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact NanoJ-Fluidics is now the basis for commercial technology in development by Abbelight 
URL https://www.biorxiv.org/content/10.1101/320416v1
 
Title NanoJ-SQUIRREL 
Description NanoJ-SQUIRREL is an analytical approach for quantifying image quality in super-resolution microscopy, provided as a GPU-enabled open-source ImageJ plugin. SQUIRREL requires two input images - a super-resolution image (or image stack) and the diffraction-limited equivalent of the same imaging volume. It then calculates an error-map, highlighting areas of the super-resolution image which exhibit poor agreement with the diffraction-limited image, and quality metrics for the super-resolution image. 
Type Of Technology Software 
Year Produced 2018 
Open Source License? Yes  
Impact NanoJ-SQUIRREL provides the technological basis to develop artificial intelligent self-driven microscopes. This is serving as a nucleator for collaborations with industry. 
URL https://bitbucket.org/rhenriqueslab/nanoj-squirrel/wiki/Home
 
Title NanoJ-SRRF (Super-Resolution Radial Fluctuations) 
Description SRRF (pronounced as surf) is as a novel open-source and high-performance analytical approach for Live-cell Super-Resolution Microscopy, provided as a fast GPU-enabled ImageJ plugin. SRRF is capable of extracting high-fidelity super-resolution information in modern microscopes (TIRF, widefield and confocals) using conventional fluorophores such as GFP. Compared to other methods, SRRF is capable of live-cell imaging over timescales ranging from minutes to hours, using sample illumination orders of magnitude lower than methods such as PALM, STORM or STED. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact SRRF is a novel super-resolution analytical approach, since its publication it started being used by thousands of researchers (>50K uses since July 2016, as tracked by Google Analytics). There are now multiple laboratories publishing work that takes advantage of this software. 
URL https://bitbucket.org/rhenriqueslab/nanoj-srrf/wiki/Home
 
Title NanoJ-VirusMapper 
Description VirusMapper is a high-throughput, open-source ImageJ plugin for single-particle analysis in fluorescence microscopy, particularly super-resolution microscopy. It provides a platform for automatic statistical mapping of conserved multi-molecular structures, such as viral substructures or intact viruses. VirusMapper uses naive averaging to create models of the distribution of particular labelled species within larger structures. 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact This software is helping create the first multi-molecular structural maps of pox-like viruses and is currently being used by multiple laboratories to model the structure of cellular supramolecular complexes. 
URL https://bitbucket.org/rhenriqueslab/nanoj-virusmapper/wiki/Home
 
Description Host laboratory for In2ScienceUK 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact We are a volunteer laboratory for the In2ScienceUK programme, an award winning initiative which empowers students from disadvantaged backgrounds to achieve their potential and progress to STEM and research careers through high quality work placements and careers guidance
Year(s) Of Engagement Activity 2016,2017,2018
URL http://in2scienceuk.org/
 
Description London Super-Resolution Club Meetings 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I am one of the main organisers of the "London Super-Resolution Group" (LSR), a bi-monthly series of highly successful meetings bringing biologists, physicist, chemists and mathematicians together - showcasing their research with Super-Resolution Microscopy. This meeting series is designed to mix UK researchers from student to group leader level and high-profile speakers (such as the recently hosted Nobel Laureate Dr. Eric Betzig).
Year(s) Of Engagement Activity 2013,2014,2015,2016
URL https://www.ucl.ac.uk/super-resolution/events/UCLSuperResolutionSymposium2015/Home
 
Description UCL Super-Resolution Symposium 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The "UCL Super-Resolution Symposium" was an international meeting on super-resolution microscopy with free registration. It has showcased cutting-edge multi-disciplinary research in the field through seminal talks, including one by the Nobel Prize Laureate Dr. Eric Betzig. This symposium also celebrated the inauguration of the super-resolution facility at UCL nucleated by an MRC Next Generation Optical Microscopy award.
Year(s) Of Engagement Activity 2015
URL https://www.ucl.ac.uk/super-resolution/events/UCLSuperResolutionSymposium2015/Home