Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT

DNA-PAINT's imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging. Hundred-fold-faster DNA-PAINT imaging is enabled by the introduction of concatenated, periodic DNA sequence motifs in the docking strand. Six orthogonal sequences are described for speed-optimized and highly multiplexed cellular imaging.