Reductively Caged, Photoactivatable DNA-PAINT for High-Throughput Super-resolution Microscopy

Area of Science:

• Biophysics
• Nanotechnology
• Microscopy

Background:

• DNA points accumulation in nanoscale topography (DNA-PAINT) achieves sub-10-nm resolution in single-molecule localization microscopy.
• High background from unbound fluorescent probes limits DNA-PAINT imaging speed and throughput.

Purpose of the Study:

• To reduce background noise in DNA-PAINT.
• To enhance imaging speed and throughput for super-resolution microscopy.

Main Methods:

• Reductive caging of fluorescent DNA probes conjugated with a cyanine dye to hydrocyanine.
• Utilizing a photoactivatable dark state for selective probe activation.
• Employing total internal reflection (TIR) illumination at 405 nm for probe activation.
• Implementing high-density analysis for increased imaging speed.

Main Results:

• Reductive caging created a photoactivatable dark state, lowering fluorescent background.
• Optically selective activation by TIR illumination preserved image quality.
• Localization density and imaging speed were increased.
• Imaging speed of conventional DNA-PAINT was enhanced by two orders of magnitude.

Conclusions:

• Reductive caging is a viable strategy to overcome background limitations in DNA-PAINT.
• This method enables high-throughput super-resolution imaging.
• The approach significantly improves the speed and efficiency of DNA-PAINT.