Spectroscopic DNA-PAINT for Simultaneous Multiplexed Super-Resolution Microscopy.

Simultaneous multiplexed super-resolution imaging remains a central challenge in single-molecule localization microscopy (SMLM), due to the limited number of spectrally distinguishable fluorophores and the tradeoff between spatial localization and spectral uncertainties. Here, we introduce spectroscopic DNA-PAINT (sDNA-PAINT), a framework that integrates DNA-PAINT with spectroscopic SMLM (sSMLM) to enable simultaneous multiplexed imaging with high spatial and spectral fidelity. Using DNA origami nanorulers, in vitro and fixed-cell imaging, we show that sDNA-PAINT conditions significantly reduce spectral uncertainty and enhance 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 four-color subcellular imaging, sDNA-PAINT achieves accurate spatial reconstruction and high classification accuracy within a single acquisition. sDNA-PAINT provides a pathway toward high-fidelity simultaneous multiplexed super-resolution interaction imaging.