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Super-Resolution Imaging and Shared Management: A Protocol for Confocal Microscopy with Multiplex Detection
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Multicolor Super-resolution Fluorescence Imaging of Cells by Expansion Microscopy.

Danush Taban1, Marvin Jungblut2, Patrick Eiring1

  • 1Department of Biotechnology & Biophysics, Biocenter, University of Würzburg, Würzburg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2026
PubMed
Summary

This study details an accelerated expansion microscopy protocol to visualize nuclear pore complex organization in HeLa cells. The method enhances experimental efficiency for super-resolution imaging of cellular structures.

Keywords:
Expansion microscopyMulticolor fluorescence imagingNuclear pore complexSuper-resolution microscopy

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Molecular Imaging

Background:

  • The nuclear pore complex (NPC) is crucial for nucleocytoplasmic transport.
  • Understanding NPC organization requires high-resolution imaging techniques.
  • Existing methods may lack efficiency or throughput for detailed structural analysis.

Purpose of the Study:

  • To present a step-by-step protocol for resolving NPC organization using expansion microscopy (ExM).
  • To introduce an accelerated ExM protocol for improved experimental efficiency and throughput.
  • To enable multicolor super-resolution imaging of the NPC.

Main Methods:

  • Utilized eight-fold expansion microscopy (ExM) on HeLa-NUP107-GFP cells.
  • Implemented pre-expansion staining with wheat germ agglutinin (WGA).
  • Employed post-digestion staining with nanobodies against GFP.
  • Performed multicolor super-resolution imaging using confocal microscopy.

Main Results:

  • Successfully resolved the organization of the nuclear pore complex (NPC).
  • Achieved approximately 30 nm spatial resolution.
  • Demonstrated an accelerated protocol enhancing experimental throughput.

Conclusions:

  • The developed accelerated ExM protocol is effective for high-resolution NPC visualization.
  • This method allows for rapid iteration and improved experimental efficiency.
  • The protocol facilitates multicolor super-resolution imaging of cellular ultrastructure.