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.