Pushing Optical Resolution to the Few-Nanometer Scale via dSTORM Imaging of Expanded Specimen-Gel Composites

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Direct stochastic optical reconstruction microscopy (dSTORM) offers nanoscale resolution (10-20 nm) but struggles with ultrastructural detail.
• Expansion microscopy (ExM) combined with dSTORM can achieve sub-10 nm resolution, but is limited by expansion factors and technical challenges with high-fold expansion.

Purpose of the Study:

• To develop a robust and compatible workflow integrating high-fold expansion microscopy with dSTORM for ultrastructural imaging.
• To overcome technical limitations in imaging highly expanded specimens for enhanced resolution.

Main Methods:

• Introduced post-labeling ten-fold robust expansion microscopy (plTREx) for enhanced hydrogel mechanical stability and fluorescence signal density.
• Optimized a re-embedding protocol to integrate plTREx with dSTORM, preventing gel shrinkage.
• Developed a workflow termed plTREx-dSTORM for compatible widefield and dSTORM imaging.

Main Results:

• Achieved imaging resolutions below 10 nm, enabling ultrastructural interpretation of cellular proteins.
• Demonstrated enhanced mechanical stability of the expansion hydrogel.
• Improved fluorescence signal density for both widefield and dSTORM imaging platforms.

Conclusions:

• plTREx-dSTORM effectively bridges the resolution gap between electron and optical microscopy.
• This integrated approach provides highly refined imaging capabilities for cellular ultrastructure.
• The developed workflow facilitates advanced nanoscale imaging of biological specimens.