Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 31, Pages 11298-11303Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0402155101
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Funding
- NIGMS NIH HHS [GM067961, R01 GM067961] Funding Source: Medline
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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 (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.
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