Nanometer-localized multiple single-molecule fluorescence microscopy

TLDR

Single‑molecule localization achieves nanometer precision by fitting the point spread function, but this approach fails when multiple fluorophores occupy a diffraction‑limited spot. The study introduces nanometer‑localized multiple‑single‑molecule (NALMS) fluorescence microscopy that combines centroid localization with photobleaching to resolve several fluorophores within a diffraction‑limited spot. NALMS employs short duplex DNA strands as nanoscale rulers, applying centroid localization and sequential photobleaching to distinguish individual fluorophores. The method achieves nanometer accuracy for two to five molecules in a diffraction‑limited area, bridging the <10 nm FRET and >100 nm diffraction limits, and enables DNA mapping at 30‑base (≈10 nm) resolution.

Abstract

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale “rulers” to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (&lt;10 nm) and diffraction-limited microscopy (&gt;100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with &lt;10-nm (i.e., 30-base) resolution is demonstrated.