Unravelling chemical and biological processes with advanced probes and enhanced resolution

基于单分子定位显微镜（SMLM）的超分辨率荧光方法正在彻底改变我们对生物学、化学和物理学的理解。矛盾的是，对于成功的SMLM成像，需要高荧光团密度来提高分辨率，但一次只能定位衍射极限点内的一个荧光团。解决这一矛盾的办法在于设计出在开、关状态之间循环的荧光团。因此，在给定的时间内，只有一部分探针被记录并成功地定位。叠加所有的定位重建图像。为了解决上述矛盾，我们提出了创新的想法，在新的和现有的探针中引导光物理/光化学途径，以确保受控循环和提高超分辨率。我们还建议不仅要开发定位信标（标记并报告感兴趣底物位置的探针），还要开发真正的反应性探针。这些探针在响应化学线索（例如，存在活性氧）时激活，导致“化学耀斑”。成像荧光团化学激活将扩大SMLM在二维和三维化学动力学中的应用，这是我们建议在细胞系统中探索的东西。接下来，我们建议使用SMLM构建基于DNA的可驱动生物相容性纳米结构。这些结构将与SMLM结合，构成生物物理工具，用于探索细胞系统中的相分离（液-液相分离和无膜细胞器的形成）等新兴概念。我们的研究是一种多层次、多方面的方法，主要有三个目标：一、揭示创新荧光成像方法的基本光过程。2。利用超分辨率成像和探针来绘制和破译细胞的氧化还原化学。3。提供了指导下一代可驱动dna纳米材料组装的规则，以及成像和探测细胞的新工具。我们的愿景是，通过明智地应用化学原理，我们将为荧光成像的变革进步做出贡献。反过来，利用革命性的荧光方法，我们可以在尚未探索的维度上解开化学过程。我们对荧光团控制和光稳定的机理见解将转化为多色成像中无与伦比的分辨率。基于可激活致敏剂的治疗策略将会出现。我们的多学科研究方法将为构建复杂的化学系统提供指导规则。它也将提供独特的方法来调和氧化还原化学与细胞的生物学。提出的工作将转化为广泛的应用，包括纳米材料、诊断和成像，从而实现生物技术、材料和生物学的颠覆性创新。这些领域的进步将对加拿大的社会和经济福祉产生积极影响。

Super resolution fluorescence methods based on single molecule localization microscopy (SMLM) are revolutionizing our understanding of biology, chemistry, and physics. Paradoxically, for successful SMLM imaging, high fluorophore density is required for improved resolution, yet only one fluorophore within a diffraction limited spot can be localized at a time. A solution to this paradox has relied in devising fluorophores that cycle between on states and off states. Only a subset of probes is thus recorded and successfully localized at a given time. Images are reconstructed superposing all the localizations. To address the above paradox, we propose innovative ideas to steer photophysical/photochemical pathways in new and existing probes, to ensure controlled cycling and improved super resolution. We also propose developing not just positional beacons (probes that are tagged to and report on the location of substrates of interest) but truly reactive probes. These are probes that activate in response to a chemical cue (e.g. presence of a reactive oxygen species) resulting in "chemical flares". Imaging fluorophore chemo-activation will expand the utility of SMLM to visualize chemical dynamics in 2D and 3D, something we propose to explore in cellular systems. We next propose to use SMLM to build actuatable biocompatible nanostructures based on DNA. These structures will constitute, coupled to SMLM, biophysical tools to explore emerging concepts such as phase segregation in cellular systems (liquid-liquid phase separation and formation of membraneless organelles). Ours is a multi-layered, multifaceted approach articulated along three goals: I. Unravelling fundamental photoprocesses toward innovative fluorescence imaging methodologies. II. Exploiting super resolution imaging and probes to map and decipher the redox chemistry of the cell. III. Providing rules to guide the assembly of next-generation actuatable DNA-based nanomaterials, new tools to image and probe the cell. Our vision is that by judiciously applying chemistry principles, we will contribute transformative advances in fluorescence imaging. In turn, upon exploiting revolutionary fluorescence methodologies, we may unravel chemical processes in, yet, unexplored dimensions. Our mechanistic insights on fluorophore control and photostabilization will translate to unsurpassed resolution in multicolor imaging. Theranostic strategies based on activatable sensitizers will emerge. Our multidisciplinary research approach will provide guiding rules toward constructing complex chemical systems. It will also render unique approaches toward reconciling the redox chemistry with the biology of the cell. The work proposed will translate to a wide range of applications, including nanomaterials, diagnostics and imaging enabling disruptive innovations in biotechnology, materials, and biology to occur. Progress in these areas will positively impact Canada's social and economic wellbeing.