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

显微镜一直是细胞生物学的主要工具。它在16世纪的出现引发了第一波“发现浪潮”--发现和理解细胞及其内部结构。然而，现代光学显微镜的基本限制（例如宽视场和共焦）使我们无法准确分辨小于300 nm的结构。花了三个世纪的时间才实现了第二次“发现浪潮”-电子显微镜（EM）的发展能够绕过这个分辨率限制，为小型生物复合体提供了新的视角，例如内吞囊泡和病毒。然而，EM有两个主要的限制，因为它不允许：1）成像活细胞和2）使用荧光标记的分子。我们现在处于超分辨率光学显微镜发展带来的“第三波发现”的最前沿-一系列方法接近EM的分辨率精度，但具有活细胞成像和分子特异性标记的能力。然而，超分辨率成像并不简单，只能通过三个关键组件之间的精细平衡来实现：1）高灵敏度的定制显微镜; 2）优化的荧光标记; 3）先进的计算分析。到目前为止，研究界对这三个因素的开发还没有完全耦合-例如，我们已经达到了一个阶段，计算机处理和硬件已经正式用于视频速率高速超分辨率，但仍然缺乏合适的无毒荧光探针。本项目旨在解决这一问题，将1）新一代具有可调光开关动力学的小探针，设计用于高速低毒性超分辨率成像; 2）高速成像系统，能够通过实时调整探针特性来修改成像微环境; 3）超分辨率采集软件能够做出数据驱动的决策，以最佳地平衡探针的光动力学，从而获得最佳的速度和分辨率。最近，我们已经开发了一种称为Super-Beacon的新型探针原型。其结构特性允许将几乎任何合成荧光团转化为具有可调节光动力学的高性能探针。基于Super-Bearing的原理，我们的目标是设计新一代针对高速多色超分辨率显微镜进行优化的探针。与此同时，我们将开发一种新的分析（软件）和成像（光学硬件）框架-称为Beacon-STORM（BeaST）-利用Super-Beacon来提高超分辨率显微镜的分辨率，速度和低光损伤。为了保持我们为研究界提供超分辨率成像关键工具的记录，我们将遵循开放获取政策，为研究人员提供工具和框架，以便他们轻松地适应和使用Super-Beasting和BeaST进行自己的研究。作为这两种高度互补技术应用的原理证明，我们将针对真核细胞生物学中的一个基本的和开放的问题--什么是细胞膜上受体的触发和所需的结构重塑，以促进网格蛋白介导的内吞作用？使用病毒样颗粒作为模型货物，我们将解决这个问题，通过超解析在体内的纳米结构的早期内吞网站，映射参与囊泡形成的细胞因子。这个问题只能通过一种方法来最佳地回答，例如所提出的方法，因为它需要一种能够在活细胞中以最小的行为干扰来解决囊泡形成位点纳米组织的系统。了解这种相互作用对于揭示内吞作用的基础和了解细胞如何处理信号噪声（如随机受体聚集）至关重要。

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.