Development of a Versatile Multiplexing Nanoscopy Platform for Cell Biology

项目摘要 了解细胞功能与用显微镜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，神经系统疾病， 纤毛病和微生物发病机制。超分辨率显微镜（SRM）在这里有潜力，因为它连接了 光学和电子显微镜之间的差距，并提供分子特异性。然而，SRM主要提供 只有几个颜色通道。这就阻碍了细胞器的全面结构图，因为许多细胞器是 多形的，并存在于多种状态，这取决于细胞内和细胞外的线索，使组合 数据集，每个显示不同的标签子集，困难。DNA-PAINT的SRM技术允许， 原理，强大的多路复用图像10个或更多的标签在一个样本，但在速度，成本和易用性的障碍 限制了它的应用。所需要的是一种高度通用的多路复用策略， 细胞器在四个关键领域有数量级的改进：获取速度， 多重探针组、空间分辨率和成本。这需要新的探头、仪器、增强的 分析和生物学验证。我们将通过三个具体目标来完成这些任务： 开发新的多功能DNA-PAINT探针，这些探针既具有荧光性，又提供快速，适应性强， 用于多路复用的低成本框架，2）用于多路复用DNA-PAINT数据的自动化采集的新平台 和分析，以“连接点”的单分子定位点在三维空间，从而创建 细胞器的膜表征，以及3）多重DNA-PAINT '细胞器的发展 模块”，以在现实的生物条件下验证这项技术，并降低进入障碍， 未来的生物用户实现这些目标及其具体成果将对使用 和SRM的可访问性来加速生物发现。

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.