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

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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.