Sub-10-nm distance measurements between fluorophores using photon-accumulation enhanced reconstruction (PACER).

Single-molecule localization microscopy (SMLM) precisely localizes individual fluorescent molecules within the wide field of view. 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, we can specifically distinguish and identify individual fluorophore, 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 showed the feasibility of PACER by resolving quantum dots that were 6.1 nm apart with 1.7-nm localization precision. Next, a Monte Carlo simulation is used to investigate the success probability of 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 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 field of view.