Unravelling chemical and biological processes with advanced probes and enhanced resolution

利用先进的探针和增强的分辨率揭示化学和生物过程

• 批准号: RGPIN-2019-05935
• 负责人: Cosa, Gonzalo
• 金额: $ 7.65万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=692894
• 关键词: Unravelling; chemical; biological; processes; advanced

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.**

基于单分子定位显微镜（SMLM）的超级分辨率荧光方法正在改变我们对生物学，化学和物理学的理解。矛盾的是，对于成功的SMLM成像，改善分辨率需要高荧光团密度，但一次可以定位在衍射有限斑点内的荧光团。解决该悖论的解决方案一直依赖于设计在状态和状态下循环的荧光团。因此，仅记录并在给定时间成功地定位了一部分问题。图像是重建所有本地化的重构。 ***为了解决上述悖论，我们提出了创新的想法，以在新问题和现有问题中引导光物理/光化学途径，以确保受控的循环和改进的超级分辨率。我们还建议不仅开发位置信标（标记为探针并报告了感兴趣的基材的位置），而且还真正反应性问题。这些问题是响应化学提示而激活的问题（例如，有活性氧的存在）导致“化学耀斑”。成像荧光团化学激活将扩大SMLM在2D和3D中可视化化学动力学的实用性，这是我们在细胞系统中探索的。我们下一个建议使用SMLM建立基于DNA的主动生物相容性纳米结构。这些结构将构成与SMLM的生物物理工具相结合的，以探索新兴概念，例如细胞系统中的相位分​​离（液 - 液相分离和膜无细胞器的形成）。 ***我们的是一种沿着三个目标表达的多层多面方法：I。将基本的光程序脱离创新的荧光成像方法。 ii。利用超级分辨率成像和问题来绘制和破译细胞的氧化还原化学。 iii。提供规则来指导下一代基于DNA的纳米材料的组装，图像和探测细胞的新工具。我们的愿景是，通过明智地采用化学原则，我们将在荧光成像中贡献变革性的进步。反过来，在利用革命性的荧光方法时，我们可能会在未探索的尺寸中阐明化学过程。基于可激活传感器的治疗策略将出现。我们的多学科研究方法将为建造复杂的化学系统提供指导规则。它还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，材料和生物学的破坏性创新。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。**