The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes

Area of Science:

• Super-resolution microscopy
• Biophysics
• Molecular imaging

Background:

• DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique relying on transient DNA hybridization for single-molecule blinking.
• The choice of fluorescent dye significantly impacts DNA-PAINT measurement quality, yet systematic performance overviews are lacking.

Purpose of the Study:

• To systematically evaluate the performance of 18 fluorescent dyes for DNA-PAINT across different spectral regions.
• To establish guidelines for selecting optimal dyes based on key performance parameters.
• To demonstrate the utility of optimized dye selection for multi-color super-resolution imaging in biological samples.

Main Methods:

• Defined and measured four key dye performance parameters: brightness, signal-to-background ratio, docking site damage, and off-target signal.
• Analyzed dyes using DNA origami nanostructures as a reference and in cellular environments targeting the nuclear pore complex.
• Performed simultaneous three-color DNA-PAINT with Exchange-PAINT for high-resolution imaging of neuronal proteins.

Main Results:

• Identified specific dyes demonstrating superior performance in brightness, signal-to-background ratio, and minimal sample damage for DNA-PAINT.
• Established a comprehensive performance overview of commonly used fluorescent dyes across different spectral regions.
• Achieved ~16 nm resolution imaging of six protein targets in neurons within 2 hours using optimized multi-color DNA-PAINT.

Conclusions:

• Dye selection is critical for maximizing DNA-PAINT performance and achieving high-quality super-resolution images.
• The study provides essential guidelines and a performance benchmark for fluorescent dye selection in DNA-PAINT.
• Optimized dye selection enables efficient, high-resolution multi-color imaging of complex biological structures.