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.

光谱单分子定位显微镜（sSMLM）首次实现了单分子的同时成像和光谱分析。当前的 sSMLM 从根本上受到光子预算减少的影响，因为来自单个随机发射的光子被分为空间通道和光谱通道。因此，空间定位和光谱分析仅使用总光子的一部分，导致两个通道的精度降低。为了提高空间和光谱精度，我们提出了对称分散的 sSMLM（或 SDsSMLM），以充分利用空间和光谱通道中单个随机发射的所有光子。 SDsSMLM 在总光子预算为 1000 的情况下实现了 10 nm 空间精度和 0.8 nm 光谱精度。与现有使用空间通道和光谱通道之间 1:3 分光比的 sSMLM 相比，SDsSMLM 将空间精度和光谱精度分别提高了 42% 和 10 %，分别是在相同的光子预算下。我们还演示了使用 SDsSMLM 的固定细胞的多色成像和三维单粒子跟踪。 SDsSMLM 可在更广泛的细胞生物学和材料科学应用中实现更精确的光谱单分子分析。