Using Dark Dyes for Fast Super-Resolution Imaging: A Proof-of-Concept Study.

Area of Science:

• Nanoscopy
• Molecular Imaging
• Biophysics

Background:

• DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) enables super-resolution imaging via transient DNA strand hybridization.
• Limitations include high background noise and slow image acquisition due to unbound imager strands.

Purpose of the Study:

• To develop and validate a novel DNA-PAINT technique utilizing intermolecular dark resonance energy transfer (DRET).
• To overcome limitations of conventional DNA-PAINT, enabling faster and lower-background super-resolution imaging.

Main Methods:

• Incorporation of a fluorenyl nucleobase (X) substitute as a dark donor into the imager strand.
• Utilizing DRET to activate fluorescence of an acceptor (ATTO 647N) on the docking strand upon hybridization.
• Single-molecule experiments to validate imager strand performance and hybridization kinetics.

Main Results:

• An 11-nucleotide (nt) X-labeled imager strand demonstrated suitable hybridization rates with the ATTO 647N-labeled docking strand.
• The DRET-based method achieved low background noise, allowing high imager strand concentrations.
• Super-resolved images of cellular microtubules were acquired within 30 seconds.

Conclusions:

• This study presents the first proof-of-concept for a DRET-based approach to fast DNA-PAINT nanoscopy.
• The novel method offers a promising route to accelerate super-resolution imaging with reduced background.
• This technique has the potential to significantly improve the speed and efficiency of nanoscale imaging in biological systems.