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

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.

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.

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.