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PHYSICAL SCIENCES / BIOLOGICAL SCIENCES / ENGINEERING / BIOPHYSICS
Nanometer-scale mapping and single-molecule detection with color-coded nanoparticle probes

Amit Agrawal^*, Rajesh Deo, Geoffrey D. Wang^*, May D. Wang^,, and Shuming Nie^*,

*Departments of Biomedical Engineering and Chemistry, Emory University and Georgia Institute of Technology, 101 Woodruff Circle, Suite 2001, Atlanta, GA 30322; Department of Physics and Astronomy, Georgia State University, Atlanta, GA 30303; and Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, VA Whitaker Building 4106, Atlanta, GA 30332

Communicated by Mostafa A. El-Sayed, Georgia Institute of Technology, Atlanta, GA, December 29, 2007 (received for review April 26, 2007)

We report a method for single-molecule detection and biomolecular structural mapping based on dual-color imaging and automated colocalization of bioconjugated nanoparticle probes at nanometer precision. In comparison with organic dyes and fluorescent proteins, nanoparticle probes such as fluorescence energy-transfer nanobeads and quantum dots provide significant advantages in signal brightness, photostability, and multicolor-light emission. As a result, we have achieved routine two-color superresolution imaging and single-molecule detection with standard fluorescence microscopes and inexpensive digital color cameras. By using green and red nanoparticles to simultaneously recognize two binding sites on a single target, individual biomolecules such as nucleic acids are detected and identified without target amplification or probe/target separation. We also demonstrate that a powerful astrophysical method (originally developed to analyze crowded stellar fields) can be used for automated and rapid statistical analysis of nanoparticle colocalization signals. The ability to rapidly localize bright nanoparticle probes at nanometer precision has implications not only for ultrasensitive medical detection but also for structural mapping of molecular complexes in which individual components are tagged with color-coded nanoparticles.

colocalization | multicolor | superresolution

Author contributions: A.A., M.D.W., and S.N. designed research; A.A. performed research; R.D., G.D.W., and M.D.W. contributed new reagents/analytic tools; A.A., G.D.W., M.D.W., and S.N. analyzed data; and A.A., M.D.W., and S.N. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0712351105/DC1.

To whom correspondence may be addressed. E-mail: maywang{at}bme.gatech.edu or snie{at}emory.edu

© 2008 by The National Academy of Sciences of the USA

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