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

Enya S. Berrevoets, Laurell F. Kessler, Ashwin Balakrishnan, Ellen Kazumi Okuda, Michaela Müller-McNicoll, Bernd Rieger (), Sjoerd Stallinga () and Mike Heilemann ()
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Enya S. Berrevoets: Delft University of Technology, Department of Imaging Physics
Laurell F. Kessler: Goethe University, Institute of Physical and Theoretical Chemistry
Ashwin Balakrishnan: Goethe University, Institute of Physical and Theoretical Chemistry
Ellen Kazumi Okuda: Goethe University, Institute of Molecular Biosciences
Michaela Müller-McNicoll: Goethe University, Institute of Molecular Biosciences
Bernd Rieger: Delft University of Technology, Department of Imaging Physics
Sjoerd Stallinga: Delft University of Technology, Department of Imaging Physics
Mike Heilemann: Goethe University, Institute of Physical and Theoretical Chemistry

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-65723-x

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