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

In a 2024 publication in Nature Methods, Steen et al. sought to determine optimal dyes for DNA-PAINT and identified four key performance metrics: brightness, signal-to-background ratio, off-target signals, and DNA-PAINT docking site damage. Such damage is dye dependent and photo-induced with evidence indicating that it is mainly caused by reactive oxygen species generated by the excited state of the fluorescent dye.

A selection of 18 popular dyes excited at 488 nm, 560 nm, and 640 nm were analyzed through imaging DNA origami structures and the nuclear pore complex protein Nup96. Among the tested dyes were Biotium’s green-emitting CF®488A, red-emitting CF®568, and far-red/near-IR emitting CF®640R and CF®660R. Six protein targets in neurons at ~16 nm resolution were also simultaneously imaged within two hours by combining a three-color TIRF (Total Internal Reflection Fluorescence) microscope setup with spectral separation, dichroic mirrors, and multichannel detection with Exchange-PAINT (Figure 1). For 488 nm excitation, the authors discovered that CF®488A performed the best at the four metrics with precision at near 10 nm in cells. Through this study, the authors establish a comprehensive guideline with principal metrics for dye selection for DNA-PAINT, along with a thorough examination of widely used fluorescent dyes for this technique.

Learn more about Biotium’s fluorescent CF® Dyes for super-resolution microscopy, featuring a wide variety of staining options with superior brightness, and signal-to-noise.

Full Citation: Steen, P.R., Unterauer, E.M., Masullo, L.A. et al. The DNA-PAINT palette: a comprehensive performance analysis of fluorescent dyes. Nat Methods 21, 1755–1762 (2024). https://doi.org/10.1038/s41592-024-02374-8