We developed a fully-automated smart fluorescence microscopy workflow that combines fast confocal microscopy, morphological object screening, event-triggered and targeted 3D super-resolution STED microscopy and quantitative image analysis. This workflow was tailored to search for and identify rare objects or events in cells, and to subsequently direct the full performance of the microscope towards a nanoscale characterization of these objects. Using this workflow, we measured the 3D nano-morphology of paraspeckles, a phase-separated membrane-less organelle (MLO) located in the nucleus of eukaryotic cells. Furthermore, we applied this workflow to detect cell organelle contact sites in living cells. This smart microscopy approach is resource-efficient, enables targeted and high-throughput characterization of rare objects and events, and is transferable to a large variety of cellular structures.

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.