Symmetrically-dispersed spectroscopic single-molecule localization microscopy

Abstract

Spectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.