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

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.