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次“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（如宽视场和共聚焦）使我们无法准确分辨小于300纳米的结构。人们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）的发展能够绕过这一分辨率限制，为小型生物复合体领域提供了新的视角，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。我们现在正处于“第三波发现”的前沿，这是由超分辨率光学显微镜的发展带来的——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，超分辨率成像不是微不足道的，它只能通过三个关键组件之间的良好平衡来实现：1)高灵敏度（通常是定制的）显微镜；2)优化荧光标记；3)高级计算分析。到目前为止，研究团体对这三个因素的发展还没有完全结合起来——例如，我们已经达到了一个阶段，计算机进程和硬件已经形式化了视频速率高速超分辨率，但仍然缺乏合适的无毒荧光探针。这阻碍了超分辨率显微镜作为一种广泛的活细胞成像工具的潜力。该项目解决了这一问题，通过整合开发1)新一代具有可调光电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)通过实时调整探头特性来改变成像微环境的高速成像系统；3)超分辨率采集软件，能够做出数据驱动的决策，以最佳平衡探针的光动力学，以获得最佳速度和分辨率。最近，我们制作了一种名为“超级信标”的新型探测器的原型。它的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-Beacons的原理，我们的目标是设计新一代探针，优化高速多色超分辨率显微镜。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) -它利用Super-Beacons在超分辨率显微镜中实现更高的分辨率，速度和低照片和化学损伤。与我们为研究界提供关键的超分辨率成像工具的记录保持一致，我们将遵循开放获取政策，为研究人员提供工具和框架，使他们能够轻松地适应和使用Super-Beacons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和开放的问题-细胞膜上受体的触发和所需的结构重塑是什么，以促进网格蛋白介导的内吞作用？利用病毒样颗粒作为模型货物，我们将通过超分辨体内早期内吞位点的纳米结构，绘制参与囊泡形成的细胞因子来解决这个问题。这个问题只能通过一种如所提出的方法来最佳地回答，因为它需要一种能够在活细胞中分解囊泡形成位点纳米组织的系统，并使其行为受到最小的破坏。了解这种相互作用对于揭示内吞作用的基础和理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.