Single-Molecule Localization Expansion Nanoscopy via Spontaneously Blinking Fluorophores

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.