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Published Online August 16, 2007
Science DOI: 10.1126/science.1146598

Reports

Submitted on June 18, 2007
Accepted on August 3, 2007

Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes

Mark Bates 1, Bo Huang 2, Graham T. Dempsey 3, Xiaowei Zhuang 4*

1 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
2 Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
3 Graduate program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
4 Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.; Department of Physics, Harvard University, Cambridge, MA 02138, USA.

* To whom correspondence should be addressed.
Xiaowei Zhuang , E-mail: zhuang{at}chemistry.harvard.edu

Recent advances in far-field optical nanoscopy have enabled fluorescence imaging with spatial resolution of 20 – 50 nm. Multicolor super-resolution imaging, however, remains challenging. In this report, we introduce a family of photo-switchable fluorescent probes and demonstrate multicolor stochastic optical reconstruction microscopy (STORM). Each probe consists of a photo-switchable "reporter" fluorophore that can be cycled between fluorescent and dark states, and an "activator" that facilitates photo-activation of the reporter. Combinatorial pairing of reporters and activators allows the creation of probes with many distinct colors. Iterative, color-specific activation of sparse subsets of these probes allows their localization with nanometer accuracy, enabling the construction of a super-resolution STORM image. Using this approach, we demonstrate multi-color imaging of DNA model samples and mammalian cells with 20 – 30 nm resolution. This technique will facilitate direct visualization of molecular interactions at the nanometer scale.


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