Spectroscopic single-molecule localization microscopy (sSMLM) was used to achieve simultaneous imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from a reduced photon budget because the photons from individual stochastic emissions are divided 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. Compared with the 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 multicolour imaging of fixed cells and three-dimensional single-particle tracking using SDsSMLM. SDsSMLM enables more precise spectroscopic single-molecule analysis in broader cell biology and material science applications. Researchers have refined the technique called spectroscopic single-molecule localization microscopy (sSMLM) to simultaneously locate individual molecules and also obtain spectral signatures carrying information about their molecular structures. The method, developed by Hao F. Zhang and colleagues at Northwestern University in Illinois, USA, is the first to use of all of the photons emitted by molecules to provide both spatial and spectral information. Previously the information delivered by photons in separate spatial and spectral channels could not be used in combination. The technique, called Symmetrically dispersed sSMLM, achieves a 42 percent increase in spatial precision and a 10 percent increase in spectral precision. The researchers also demonstrated the technique for multicolour imaging in cells and three-dimensional tracking for monitoring nanoparticles. It should significantly enhance spectroscopic analysis at the single-molecule level in both biology and materials science.
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