Correlative Single-Molecule FRET and DNA-PAINT Imaging

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• DNA-PAINT offers super-resolution imaging and spectrally unlimited multiplexing.
• Current DNA-PAINT multiplexing methods rely on sequential imaging, limiting throughput.
• There is a need for faster, more specific multiplexed super-resolution techniques.

Purpose of the Study:

• To combine DNA-PAINT with single-molecule Förster Resonance Energy Transfer (smFRET) for multiplexed super-resolution imaging.
• To utilize FRET efficiency as a parameter for high-specificity multitarget detection.
• To achieve accurate nanoscale distance measurements in the 1-10 nm range.

Main Methods:

• Integration of DNA-PAINT with single-molecule FRET.
• Utilizing FRET efficiency for multiplexed imaging.
• Demonstration on DNA origami structures with specifically designed binding sequences.
• Extraction of FRET values from closely spaced binding sites (~55 nm).

Main Results:

• Successful correlated single-molecule FRET and super-resolution imaging.
• Demonstration of super-resolution FRET imaging with high specificity.
• Accurate FRET value extraction from nanoscale binding sites.
• Achieved low background noise in multiplexed imaging.

Conclusions:

• The combination of FRET and DNA-PAINT enables multiplexed super-resolution imaging.
• This approach offers high specificity and low background.
• It facilitates accurate distance readout in the 1-10 nm range, advancing nanoscale imaging capabilities.