Spectroscopic DNA-PAINT for simultaneous multiplexed super-resolution microscopy

and fixed-cell imaging, we show that sDNA-PAINT conditions significantly improve spectral precision and photon budgets compared to conventional glass and antibody-conjugated conditions in sSMLM imaging. Across representative dyes from three fluorophore families (Rhodamine, Cyanine and Oxazine), narrow spectral centroid distributions are observed to enable reliable statistical discrimination at the single-molecule level, even for dyes with heavily overlapping ensemble spectra. In dual-target cellular imaging, sDNA-PAINT achieves accurate spatial reconstruction and high classification accuracy, demonstrating high-fidelity simultaneous multiplexing within a single acquisition. sDNA-PAINT provides a pathway toward high-throughput simultaneous multiplexed super-resolution interaction imaging.