A Universal Labeling Strategy for Nucleic Acids in Expansion Microscopy.

Area of Science:

• Molecular Biology
• Microscopy Techniques
• Genetics

Background:

• Expansion microscopy (ExM) allows nanoscale imaging of RNA within cells.
• Current methods for RNA labeling in ExM can be complex.
• Improved spatial resolution of gene expression patterns is needed.

Purpose of the Study:

• To develop and validate multivalent reagents for simplified RNA labeling in ExM.
• To assess the compatibility of these reagents with existing techniques like RNA FISH.
• To achieve high-resolution nanoscale imaging of RNA with ExM.

Main Methods:

• Development of multivalent reagents for DNA oligonucleotide probe attachment.
• Integration of reagents with *in situ* hybridization chain reaction RNA FISH.
• Application of reagents for multicolor staining and imaging.
• Validation of labeling strategies in ×4 and ×10 ExM.

Main Results:

• Reagents are compatible with RNA FISH and ensure transcript retention.
• Successful multicolor staining was achieved using the developed method.
• Achieved ~50 nm resolution in ×10 ExM with both pre- and postexpansion labeling.
• Demonstrated rapid functionalization of DNA oligonucleotides for ExM.

Conclusions:

• The novel multivalent molecules streamline DNA oligonucleotide functionalization for ExM.
• This approach enhances RNA imaging capabilities at the nanoscale.
• The method supports multicolor staining and high-resolution spatial transcriptomics.