Symmetrically dispersed spectroscopic single-molecule localization microscopy

Abstract

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.

Super‐resolution fluorescence microscopy‐based single‐molecule spectroscopy

“…To increase the number of detected photons, Song et al proposed a symmetrically dispersed SR-STORM method that improves the photon detection efficiency for a grating-based SR-STORM. 34 In a grating-based SR-STORM and a single-objective-based SR-STORM method, both spatial and spectral analysis only use a portion of the total photons, thus resulting in reduced precision in both channels. To overcome this limitation, Song et al fully utilized all photons from individual molecules by using two symmetrically dispersed spectral channels instead of one spatial channel and one spectral channel, thereby improving the spatial and spectral precision by 42% and 10%, respectively.…”

“…To overcome this limitation, Song et al fully utilized all photons from individual molecules by using two symmetrically dispersed spectral channels instead of one spatial channel and one spectral channel, thereby improving the spatial and spectral precision by 42% and 10%, respectively. 34 The detection efficiency for photon number is also expected to improve by developing cameras with higher sensitivity or grating with higher transmission efficiency.…”

Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

“…Consequently, spectrally resolved images are usually acquired in a point-scanning manner by dispersing the fluorescence from one single spot of the sample at a time. Alternatively, sparse single molecules may be spectrally dispersed in the wide-field to accumulate super-resolution images over many camera frames 12 , 13 . Both scenarios place substantial constraints on temporal resolution.…”

Multiplexed PSF Engineering for Three-Dimensional Multicolor Particle Tracking

“…Sensitive detectors (cameras) in fluorescence microscopy are typically monochrome; therefore, the most straightforward way to obtain spectral information is to perform sequential image acquisition, naturally entailing a decrease in temporal resolution. Alternatively, it is possible to preserve temporal resolution by sacrificing FOV, either by assigning different FOVs to different spectral channels ,, or by acquiring a designated spectral imaging channel alongside a spatial imaging channel. , This approach is photon-efficient and enables simultaneous multicolor acquisition but significantly reduces the FOV or is costly and space-consuming due to the necessity of multiple cameras …”