Expansion Stimulated Emission Depletion Microscopy (ExSTED)

Area of Science:

• Cellular and Molecular Imaging
• Biophysics
• Microscopy Techniques

Background:

• Stimulated emission depletion (STED) microscopy offers enhanced resolution for cellular ultrastructure.
• Expansion microscopy (ExM) achieves subdiffraction resolution by physically enlarging biological samples.
• Current super-resolution techniques have limitations in achieving maximal resolution.

Purpose of the Study:

• To combine ExM and STED microscopy (ExSTED) for unprecedented resolution.
• To establish a robust method for imaging cellular ultrastructure at the ten-nanometer scale.
• To identify requirements for high-fidelity labeling in ExSTED.

Main Methods:

• Integration of expansion microscopy (ExM) with stimulated emission depletion (STED) microscopy.
• Physical sample enlargement using hydrogel cross-linking and swelling.
• High-fidelity labeling strategies utilizing multi-epitopes for dense emitter distribution.

Main Results:

• Achieved a resolution increase of up to 30-fold compared to conventional microscopy (<10 nm lateral, ~50 nm isotropic).
• Demonstrated the capability to resolve ultrastructural details with the ExSTED technique.
• Identified the necessity of high-fidelity, multi-epitope labeling for optimal ExSTED performance.

Conclusions:

• The combined ExSTED approach provides a powerful template for super-resolution microscopy.
• This method enables visualization of entire cells at the ten-nanometer resolution range.
• ExSTED overcomes limitations of individual ExM and STED techniques for ultrastructural studies.