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Mapping Absolute DNA Density in Cell Nuclei using Single-molecule Localization Microscopy
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Single-Molecule Localization Expansion Nanoscopy via Spontaneously Blinking Fluorophores.

Lu Yang1, Tianli Zhai1, Song Chen1

  • 1Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials Institution, Fudan University, Shanghai 200438, China.

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Summary

We developed a new method combining expansion microscopy (ExM) and super-resolution microscopy (STORM) using a novel dye. This approach overcomes previous limitations, enabling clearer nanoscale imaging of cellular structures like mitochondria and microtubules.

Keywords:
HMSiRexpansion microscopyspontaneously blinking dyestochastic optical reconstruction microscopysuper-resolution imaging

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Biophysics

Background:

  • Expansion microscopy (ExM) and stochastic optical reconstruction microscopy (STORM) enable super-resolution imaging of cellular ultrastructures.
  • Challenges include hydrogel shrinkage and fluorophore bleaching, hindering Ex-STORM integration.

Purpose of the Study:

  • To overcome limitations in Ex-STORM by employing a novel spontaneously blinking dye.
  • To establish a streamlined workflow for enhanced nanoscale imaging of cellular ultrastructures.

Main Methods:

  • Utilized HMSiR, a silicon rhodamine dye with intrinsic stochastic blinking at physiological pH.
  • Developed a streamlined Ex-STORM workflow achieving stable linear expansion.
  • Applied the method to visualize mitochondrial ultrastructure and microtubule organization.

Main Results:

  • Achieved a stable linear expansion factor of 4.2 ± 0.3.
  • Resolved mitochondrial ultrastructure, including TOM20 protein clusters (~35 nm).
  • Visualized microtubule organization with ~55 nm widths, providing nanoscale protein distribution insights.

Conclusions:

  • HMSiR dye overcomes Ex-STORM limitations, improving fluorescence retention and eliminating specialized buffers.
  • The streamlined Ex-STORM workflow offers enhanced clarity for cellular ultrastructure imaging.
  • This approach advances high-resolution imaging in cell biology, enabling exploration of the cellular nanoworld.