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.
Single‐molecule localization microscopy (SMLM) precisely localizes individual fluorescent molecules within the wide field of view (FOV). However, the localization precision is fundamentally limited to around 20 nm due to the physical photon limit of individual stochastic single‐molecule emissions. Using spectroscopic SMLM (sSMLM) to resolve their distinct fluorescence emission spectra, individual fluorophore is specifically distinguished and identified, even the ones of the same type. Consequently, the reported photon‐accumulation enhanced reconstruction (PACER) method accumulates photons over repeated stochastic emissions from the same fluorophore to significantly improve the localization precision. This work shows the feasibility of PACER by resolving quantum dots that are 6.1 nm apart with 1.7 nm localization precision. Next, a Monte Carlo simulation is used to investigate the success probability of the PACER's classification process for distance measurements under different conditions. Finally, PACER is used to resolve and measure the lengths of DNA origami nanorulers with an inter‐molecular spacing as small as 6 nm. Notably, the demonstrated sub‐2 nm localization precision bridges the detection range between Förster resonance energy transfer (FRET) and conventional SMLM. Fully exploiting the underlying imaging capability can potentially enable high‐throughput inter‐molecular distance measurements over a large FOV.
- NSF-PAR ID:
- 10200752
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Photonics Research
- Volume:
- 1
- Issue:
- 2
- ISSN:
- 2699-9293
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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