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Improving STED microscopy with SUPPOSe: enhancing resolution from a single-image.

Micaela Toscani1, Axel M Lacapmesure1

  • 1Laboratorio de Fotónica, IIBM-FIUBA, CONICET, Buenos Aires, Argentina.

Methods and Applications in Fluorescence
|March 8, 2024
PubMed
Summary

The SUPPOSe algorithm enhances Stimulated Emission Depletion (STED) microscopy, revealing the nuclear pore complex's (NPC) eightfold symmetry. This advancement allows for reliable nanoscale object retrieval from single microscopy acquisitions.

Keywords:
STEDSUPPOSeconvolution-based algorithmenhancemicroscopynuclear pore complexsuper-resolution

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • The nuclear pore complex (NPC) is a large protein structure essential for nucleocytoplasmic transport.
  • The NPC's characteristic ring structure and eightfold symmetry are crucial for its function.
  • Stimulated Emission Depletion (STED) microscopy offers high-resolution imaging but struggles to resolve the NPC's full symmetry.

Purpose of the Study:

  • To improve the resolution limit of STED microscopy using the SUPPOSe algorithm.
  • To characterize the nanoscale structure of the nuclear pore complex (NPC).
  • To assess the reliability of the SUPPOSe algorithm in retrieving sub-resolution nanoscale objects.

Main Methods:

  • Application of the SUPPOSe algorithm to STED microscopy images of endogenously labeled Nup96 in NPCs.
  • Analysis of 562 single NPCs to determine structural parameters.
  • Probabilistic modeling to assess labeling efficiency and structural compatibility.

Main Results:

  • The SUPPOSe algorithm successfully resolved the octagonal structure of the NPC, revealing its eightfold symmetry.
  • The average NPC radius was determined to be R = 54.2 ± 2.9 nm.
  • An effective labeling efficiency of 31% was calculated, consistent with Single Molecule Localization Microscopy standards.

Conclusions:

  • The SUPPOSe algorithm significantly enhances STED microscopy capabilities for resolving complex nanoscale structures like the NPC.
  • SUPPOSe reliably retrieves sub-resolution nanoscale information from single, potentially noisy, microscopy acquisitions.
  • This method provides a powerful tool for detailed structural analysis of biological macromolecules.