Single extracellular vesicle imaging via rolling circle amplification-expansion microscopy.

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Diagnostics

Background:

• Extracellular vesicles (EVs) are crucial for diagnostics but challenging to analyze due to size, low biomarker abundance, and heterogeneity.
• Current methods struggle with sensitive, multi-omic profiling of individual EVs.

Purpose of the Study:

• To develop a super-resolution technique for multi-omic profiling of single extracellular vesicles (EVs).
• To enable sensitive detection of EV membrane proteins and miRNAs for clinical applications.

Main Methods:

• Integration of rolling circle amplification (RCA) with expansion microscopy (ExM) for super-resolution imaging.
• Utilized switch hairpin probes for membrane protein detection and EV-liposome fusion with molecular beacons for miRNA detection.
• Employed hydrogel-mediated expansion for enlarged single-EV analysis.

Main Results:

• Achieved super-resolution multi-omic profiling of single EVs using conventional fluorescence microscopy.
• Demonstrated sensitive detection of EV membrane proteins and miRNAs.
• Successfully quantified miRNA-21 levels in plasma EVs to distinguish cancer patients from healthy donors and predict immunotherapy efficacy in a cohort of 86 patients.

Conclusions:

• RCA-ExM provides a sensitive and specific method for single-EV multi-omic profiling.
• This technique shows significant promise for minimally invasive diagnostics and treatment monitoring in oncology.
• Enables advanced analysis of EV biomarkers for personalized medicine.