Orthogonally Dispersed Spectroscopic Single‐Molecule Localization Microscopy

Spectroscopic single-molecule localization microscopy (sSMLM) simultaneously acquires both spatial and spectral information from fluorescent molecules, facilitating molecular characterization analysis and multicolor imaging. However, this technique poses a fundamental dilemma: a finite photon budget must be split between localization and spectroscopy, limiting the performance of both. To alleviate this trade-off, we propose orthogonally dispersed sSMLM (ODsSMLM). By modulating single-molecule emission spectra through an orthogonal structure, ODsSMLM allows all photons to be used for both localization and spectral characterization. Crucially, this approach provides isotropic lateral localization precision, effectively removing the inherent dispersion-dependent localization artifacts of other methods. Using simulated data, we demonstrate that under a 3000-photon budget, ODsSMLM attains a localization precision of 10 nm and a spectral precision of 1.1 nm. Moreover, in dual-color imaging experiments of microtubules and clathrin, ODsSMLM achieved isotropic lateral resolution of 27 nm.