Single-Step Expansion SMLM Enables Molecular-Resolution Imaging in Cells and Isolated Proteins

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Expansion microscopy (ExM) offers nanoscale imaging but faces challenges integrating with single-molecule localization microscopy (SMLM).
• Existing methods struggle with hydrogel and photoswitching buffer incompatibility, hindering combined super-resolution imaging.

Purpose of the Study:

• To develop a streamlined ExM approach compatible with SMLM.
• To enable high-resolution imaging of biological structures using spontaneously blinking dyes in expanded samples.

Main Methods:

• A single-step ExM protocol was developed, eliminating the need for re-embedding or buffer exchange.
• The method utilizes spontaneously blinking dyes for SMLM on 6-14× expanded hydrogel samples.

Main Results:

• Achieved nanometer-scale spatial resolution in ExM-prepared samples.
• Successfully resolved the organization of the nuclear pore complex.
• Determined the molecular structure of recombinant homotrimeric proliferating cell nuclear antigen.

Conclusions:

• The novel single-step ExM-SMLM approach simplifies super-resolution imaging.
• This method enhances the study of molecular organization and structure at the nanoscale.