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Published Online January 3, 2008
Science DOI: 10.1126/science.1153529

Reports

Submitted on November 28, 2007
Accepted on December 17, 2007

Three-Dimensional Super-Resolution Imaging by Stochastic Optical Reconstruction Microscopy

Bo Huang 1, Wenqin Wang 2, Mark Bates 3, Xiaowei Zhuang 4*

1 Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
2 Department of Physics, Harvard University, Cambridge, MA 02138, USA.
3 School of Engineering and Applied Sciences, 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 fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 – 30 nm in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. Here, we demonstrate 3D stochastic optical reconstruction microscopy (STORM) by determining both axial and lateral positions of individual fluorophores with nanometer accuracy using optical astigmatism. Iterative, stochastic activation of photo-switchable probes enables high-precision 3D localization of each probe and thus the construction of a 3D image without scanning the sample. Using this approach, we achieved an image resolution of 20 – 30 nm in the lateral dimensions and 50 – 60 nm in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.


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