Shahid, M. A.; Patel, K.; Miller, D. A.; Zhang, Y.

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 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 multiple commonly used fluorophores, narrow spectral centroid distributions 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 true simultaneous multiplexing within a single acquisition. sDNA-PAINT provides a pathway toward high-throughput simultaneous multiplexed super-resolution imaging.