Spectroscopic DNA-PAINT for simultaneous multiplexed super-resolution microscopy

ABSTRACT 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 trade-off between spatial and spectral precision. 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 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. Competing Interest Statement The authors have declared no competing interest.