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Published online on July 26, 2004, 10.1073/pnas.0402155101
PNAS | August 3, 2004 | vol. 101 | no. 31 | 11298-11303


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BIOPHYSICS
Nanometer-localized multiple single-molecule fluorescence microscopy

Xiaohui Qu *, {dagger}, David Wu {dagger}, {ddagger} §, Laurens Mets ¶, and Norbert F. Scherer {dagger}, {ddagger}, ||

Departments of *Physics, Molecular Genetics and Cell Biology, and {ddagger}Chemistry and {dagger}Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 23, 2004 (received for review March 26, 2004)

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.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: NALMS, nanometer-localized multiple single-molecule; FRET, fluorescence resonance energy transfer; PNA, peptide nucleic acid; TIRF, total internal reflection fluorescence; CCD, charge-coupled device.

Note. While this paper was under review, a paper appeared (27) reporting the use of photobleaching to resolve two fluorophores within the diffraction limit. Our work extends the concept to the nanometer-precision localization of multiple fluorophores that are unresolved in a static image. The image processing used in the present paper differs from that reported in ref. 27. These differences in numerical methods may affect the precision of fluorophore localization in certain circumstances and also the computation time for analyzing the images, particularly when multiple fluorophores are involved. As shown in this work, ultraresolution of multiple fluorophores will be important in many applications, including DNA mapping.

§ Present address: David Geffen School of Medicine, University of California, Los Angeles, CA 90095.

|| To whom correspondence should be addressed at: 5735 South Ellis Avenue, SCL 033, Chicago, IL 60637. E-mail: nfschere{at}uchicago.edu.

© 2004 by The National Academy of Sciences of the USA


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