Spectrally Resolved and Functional Super-resolution Microscopy via Ultrahigh-Throughput Single-Molecule Spectroscopy

Area of Science:

• Optical Microscopy
• Spectroscopy
• Biophysics
• Chemical Physics

Background:

• Single-molecule localization microscopy (SMLM) offers high spatial resolution (~10 nm) but lacks spectral information.
• Existing methods for spectral mapping of single molecules are low-throughput and unsuitable for dense samples.
• Integrating spectral data with SMLM is crucial for advanced molecular analysis.

Purpose of the Study:

• To develop spectrally resolved SMLM (SR-SMLM) for high-throughput spectral and spatial analysis of single molecules.
• To introduce functional SRM (f-SRM) for mapping spatiotemporal physicochemical parameters.
• To demonstrate the application of these techniques in biological and chemical systems.

Main Methods:

• Developed a wide-field spectral measurement scheme combined with SMLM's on-off switching.
• Applied SR-SMLM to record spectra and positions of millions of single molecules synchronously.
• Utilized environment-sensing probes for f-SRM to encode functional information into spectral responses.

Main Results:

• Achieved ultrahigh-throughput single-molecule spectroscopy and SR-SMLM, enabling multiplexed imaging of spectrally similar dyes.
• Demonstrated SR-SMLM's ability to analyze single-molecule reaction pathways with high throughput.
• Revealed nanoscale membrane heterogeneity in live mammalian cells using f-SRM and Nile Red, correlating polarity with cholesterol levels.

Conclusions:

• SR-SMLM and f-SRM significantly enhance super-resolution microscopy by adding spectral and functional dimensions.
• These techniques provide new avenues for probing biological and chemical systems at the single-molecule and nanoscale levels.
• The integration allows for unprecedented detail in understanding molecular interactions and cellular environments.