Spectroscopic single-molecule localization microscopy (sSMLM) generates super-resolution images of single molecules while simultaneously capturing the spectra of their fluorescence emissions. However, sSMLM splits photons from single-molecule emissions into a spatial channel and a spectral channel, reducing both channels’ precisions. It is also challenging in transmission grating-based sSMLM to achieve a large field-of-view (FOV) and avoid overlap between the spatial and spectral channels. The challenge in FOV has further significance in single-molecule tracking applications. In this work, we analyzed the correlation between the spatial and spectral channels in sSMLM to improve its spatial precision, and we developed a split-mirror assembly to enlarge its FOV. We demonstrate the benefits of these improvements by tracking quantum dots. We also show that we can reduce particle-identification ambiguity by tagging each particle with its unique spectral characteristics.
Single-molecule localization microscopy (SMLM) breaks the optical diffraction limit by numerically localizing sparse fluorescence emitters to achieve super-resolution imaging. Spectroscopic SMLM or sSMLM further allows simultaneous spectroscopy and super-resolution imaging of fluorescence molecules. Hence, sSMLM can extract spectral features with single-molecule sensitivity, higher precision, and higher multiplexity than traditional multicolor microscopy modalities. These new capabilities enabled advanced multiplexed and functional cellular imaging applications. While sSMLM suffers from reduced spatial precision compared to conventional SMLM due to splitting photons to form spatial and spectral images, several methods have been reported to mitigate these weaknesses through innovative optical design and image processing techniques. This review summarizes the recent progress in sSMLM, its applications, and our perspective on future work.
- Award ID(s):
- 1954430
- NSF-PAR ID:
- 10402590
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Nano Convergence
- Volume:
- 10
- Issue:
- 1
- ISSN:
- 2196-5404
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.
