Fluorogenic speed-optimized DNA-PAINT probes enable super-resolution imaging of whole cells

Area of Science:

• Biophysics
• Molecular Imaging
• Nanotechnology

Background:

• DNA-PAINT super-resolution microscopy offers molecular-scale, multiplexed, and quantitative imaging.
• Current limitations include slow probe binding kinetics and high background noise, hindering throughput.
• Existing speed-optimized probes face design constraints due to a binding-quenching tradeoff.

Purpose of the Study:

• To introduce a novel modular probe architecture for DNA-PAINT.
• To decouple probe binding kinetics from fluorophore-quencher interactions.
• To enhance imaging speed, signal-to-background ratio, and overall efficiency.

Main Methods:

• Designed a modular probe architecture integrating speed-optimized DNA motifs with PEG spacers.
• Utilized DNA origami nanostructures for probe assembly and characterization.
• Validated probe performance in cellular imaging of nuclear targets and endoplasmic reticulum.

Main Results:

• Demonstrated enhanced localization rates and signal-to-background ratios compared to existing probes.
• Achieved improved imaging efficiency using the new modular probe architecture.
• Successfully applied the probes for 3D imaging of cellular structures with standard widefield illumination.

Conclusions:

• The modular probe architecture effectively overcomes key limitations in DNA-PAINT super-resolution microscopy.
• This approach enables faster, more efficient, and lower-background molecular imaging.
• The developed framework supports advanced multiplexed and 3D super-resolution imaging applications.