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Originally published in Science Express on 3 January 2008
Science 8 February 2008:
Vol. 319. no. 5864, pp. 810 - 813
DOI: 10.1126/science.1153529
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Reports

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

Bo Huang,1,2 Wenqin Wang,3 Mark Bates,4 Xiaowei Zhuang1,2,3*

Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable 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 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.

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

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

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