Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy

Area of Science:

• Biophysics
• Nanotechnology
• Molecular Biology

Background:

• Fluorescence microscopy for single-molecule gene expression analysis in tissues suffers from low signal-to-noise ratios.
• Autofluorescence and background signals in tissue sections significantly hinder accurate quantification.
• Existing methods lack the sensitivity and specificity for in situ single-molecule mRNA detection in complex tissues.

Purpose of the Study:

• To develop a novel background-free method for quantifying messenger RNA (mRNA) at single-molecule resolution directly in tissues.
• To utilize second-harmonic generation (SHG) nanocrystals as probes for enhanced signal detection.
• To specifically target and quantify human epidermal growth receptor 2 (Her2) mRNA in cancer tissues.

Main Methods:

• Demonstrated coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes.
• Developed specific labeling strategies for Her2 surface marker and Her2 mRNA using BTO probes.
• Quantified Her2 mRNA at single-copy sensitivity within specific cell phenotypes in situ.

Main Results:

• Achieved a stable SHG signal from BTO nanoprobes, overcoming tissue autofluorescence limitations.
• Successfully quantified Her2 mRNA at single-molecule resolution in Her2-expressing cancer tissues.
• Demonstrated single-cell resolution for mRNA quantification directly within tissue samples.

Conclusions:

• Introduced a background-free, SHG-based nanocrystal probe strategy for sensitive mRNA detection in tissues.
• Established a proof-of-concept for a cross-platform approach enabling in situ single-cell resolution analysis.
• Paved the way for advanced molecular diagnostics and research in complex biological systems.