Smart 3D super-resolution microscopy reveals the architecture of the RNA scaffold in a nuclear body

Small subcellular organelles orchestrate key cellular functions. How biomolecules are spatially organized within these assemblies is poorly understood. Here, we report an automated super-resolution imaging and analysis workflow that integrates confocal microscopy, morphological object screening, targeted 3D super-resolution STED microscopy and quantitative image analysis. Using this smart microscopy workflow, we target the 3D organization of NEAT1, an architectural RNA that constitutes the structural backbone of paraspeckles, a membraneless nuclear organelle. Using site-specific labeling, morphological sorting and particle averaging, we reconstruct the morphological space of paraspeckles along their development cycle from over 10,000 individual particles. Applying spherical harmonics analysis, we report so-far unknown heterotypes of NEAT1 RNA organization. By integrating multi-positional labeling, we determine the coarse conformation of NEAT1 within the organelle and show that the 3’ end forms a loop-like structure at the surface of the paraspeckle. Our study reveals key structural features of paraspeckle structure and growth, as well as the molecular organization of its scaffolding RNA.