Spectroscopic single-molecule localization microscopy (sSMLM) provides simultaneous spatial and spectral information from individual emitters, but its broader application is constrained by two key factors: the limited photon budget of single fluorophores and the presence of single-molecule fluorescence spectral heterogeneity (smFLUSH). DNA-PAINT offers a unique solution by continuously replenishing fluorophores through transient binding, ensuring a sustainable photon supply for both localization and spectral readout. To enable reliable multiplexing, however, smFLUSH of the dyes must be statistically quantified. Here, we present a two-color spectroscopic DNA-PAINT framework that incorporates smFLUSH characterization of red-channel dyes to guide dye selection and spectral separation. We show that smFLUSH restricts the number of distinguishable probes within a given spectral channel, and we quantify these variations across different environments, including nanorulers and cellular targets. Finally, we demonstrate two-color spectroscopic DNA-PAINT imaging in cells, achieving improved spectral precision and multiplexing performance compared to conventional sequential acquisition. Our results highlight DNA-PAINT as a robust approach to overcome photon budget limitations in sSMLM, while smFLUSH characterization provides a pathway toward photon-efficient, multiplexed super-resolution imaging of complex biological systems.
Shahid et al. (Sun,) studied this question.