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 phot...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.展开更多
基金supported in part by NSF grants CBET-1706642 and EFMA-1830969 and NIH grants R01EY026078 and R01EY029121supported by the Christine Enroth-Cugell and David Cugell Graduate Fellowship in Biomedical Engineering and Visual Neuroscience.
文摘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.
