Development of a Versatile Multiplexing Nanoscopy Platform for Cell Biology

项目摘要 了解细胞功能与观察细胞器超微结构的能力密切相关 分子特异性，并观察它在癌症、神经疾病、 纤毛疾病和微生物发病机制。超分辨率显微镜(SRM)在这方面具有潜力，因为它架起了 光学显微镜和电子显微镜之间的间隙，并提供分子特异性。然而，SRM主要提供 只有几个颜色通道。这禁止了细胞器的全面体系结构图，因为许多细胞器都是这样 多形性，并根据细胞内和细胞外线索以多种状态存在，使得 每个数据集都显示了不同的标签子集，这是很困难的。DNA-Paint的SRM技术允许 原理上，强大的多路复用器可在一个样本中对10个或更多标签进行图像处理，但在速度、成本和易用性方面存在障碍 使用的限制了它的应用。我们需要的是一种高度通用的多路复用策略，以实现 细胞器在四个关键领域有了数量级的改进：获取速度，在 多路复用探头组、空间分辨率和成本。这需要新的探头、仪器、增强的 分析和生物验证。我们将通过三个具体目标来处理这些任务：1) 开发新的多功能DNA涂料探针，既能产生荧光，又能提供一种快速、适应性强、 低成本的多路复用框架；2)多路DNA-PAINT数据自动获取的新平台 以及分析，以在三维中将单分子定位点的点连接起来，从而创建 细胞器的膜表示；3)复合型DNA-Paint‘细胞器的发展 模块在现实的生物条件下验证这项技术，并降低进入门槛 未来的生物使用者。实现这些目标及其具体交付成果将对使用产生广泛的影响 以及SRM的可获得性，以加速生物发现。

Project Summary Understanding cellular function is intimately linked with the ability to visualize organelle ultrastructure with molecular specificity and to observe how it is altered in diseases such as cancer, neurological disease, ciliopathies and microbial pathogenesis. Super-resolution microscopy (SRM) has potential here as it bridges the gap between light and electron microscopy and provides molecular specificity. However, SRM mostly offers only a few color channels. This prohibits a comprehensive architectural map of organelles, as many are pleomorphic and exist in multiple states depending on intra- and extracellular cues, making the combination of datasets, each showing different subsets of labels, difficult. The SRM technique of DNA-PAINT allows, in principle, powerful multiplexing to image 10 or more labels in one sample, but hurdles in speed, cost and ease of use have limited its application. What is needed is a highly versatile multiplexing strategy to enable SRM of organelles with an order-of-magnitude improvement in four key areas: acquisition speed, switching between multiplex probe sets, spatial resolution, and cost. This requires new probes, instrumentation, enhanced analysis, and biological validation. We will approach these tasks through three Specific Aims: 1) the development of new versatile, DNA-PAINT probes that are both fluorogenic and provide a fast, adaptable, low-cost framework for multiplexing, 2) a new platform for automated acquisition of multiplex DNA-PAINT data and analytics to ‘connect the dots’ of single-molecule localization points in three dimensions and thereby create membrane representations of organelles, and 3) the development of multiplexed DNA-PAINT ‘organelle modules’ to validate this technology under realistic biological conditions and lower the entrance hurdle for future biological users. Achieving these aims and their concrete deliverables will have a wide impact on the use and accessibility of SRM to accelerate biological discovery.