Three-dimensional Super-resolution Imaging by Stochastic Optical Reconstruction Microscopy

Overview

Journal Science

Specialty Science

Date 2008 Jan 5

PMID 18174397

Citations 973

Authors

Bo Huang

Wenqin Wang

Mark Bates

Xiaowei Zhuang

Affiliations

Soon will be listed here.

Abstract

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.

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References

Slepnev V, De Camilli P . Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci. 2001; 1(3):161-72. DOI: 10.1038/35044540. View

Heuser J, Anderson R . Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989; 108(2):389-400. PMC: 2115439. DOI: 10.1083/jcb.108.2.389. View

Gelles J, Schnapp B, Sheetz M . Tracking kinesin-driven movements with nanometre-scale precision. Nature. 1988; 331(6155):450-3. DOI: 10.1038/331450a0. View

Barak L, Webb W . Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts. J Cell Biol. 1981; 90(3):595-604. PMC: 2111891. DOI: 10.1083/jcb.90.3.595. View

Zipfel W, Williams R, Webb W . Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol. 2003; 21(11):1369-77. DOI: 10.1038/nbt899. View

Hell S . Toward fluorescence nanoscopy. Nat Biotechnol. 2003; 21(11):1347-55. DOI: 10.1038/nbt895. View

Yildiz A, Forkey J, McKinney S, Ha T, Goldman Y, Selvin P . Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science. 2003; 300(5628):2061-5. DOI: 10.1126/science.1084398. View

Speidel M, Jonas A, Florin E . Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging. Opt Lett. 2003; 28(2):69-71. DOI: 10.1364/ol.28.000069. View

Thompson R, Larson D, Webb W . Precise nanometer localization analysis for individual fluorescent probes. Biophys J. 2002; 82(5):2775-83. PMC: 1302065. DOI: 10.1016/S0006-3495(02)75618-X. View

10.

Kao H, Verkman A . Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys J. 1994; 67(3):1291-300. PMC: 1225486. DOI: 10.1016/S0006-3495(94)80601-0. View

11.

Nagorni M, Hell S . 4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. J Struct Biol. 1999; 123(3):236-47. DOI: 10.1006/jsbi.1998.4037. View

12.

Moerner W, Orrit M . Illuminating single molecules in condensed matter. Science. 1999; 283(5408):1670-6. DOI: 10.1126/science.283.5408.1670. View

13.

Gustafsson M, Agard D, Sedat J . I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc. 1999; 195(Pt 1):10-6. DOI: 10.1046/j.1365-2818.1999.00576.x. View

14.

Chen I, Ting A . Site-specific labeling of proteins with small molecules in live cells. Curr Opin Biotechnol. 2005; 16(1):35-40. DOI: 10.1016/j.copbio.2004.12.003. View

15.

Bates M, Blosser T, Zhuang X . Short-range spectroscopic ruler based on a single-molecule optical switch. Phys Rev Lett. 2005; 94(10):108101. PMC: 2652517. DOI: 10.1103/PhysRevLett.94.108101. View

16.

Egner A, Hell S . Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 2005; 15(4):207-15. DOI: 10.1016/j.tcb.2005.02.003. View

17.

Gustafsson M . Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci U S A. 2005; 102(37):13081-6. PMC: 1201569. DOI: 10.1073/pnas.0406877102. View

18.

Giepmans B, Adams S, Ellisman M, Tsien R . The fluorescent toolbox for assessing protein location and function. Science. 2006; 312(5771):217-24. DOI: 10.1126/science.1124618. View

19.

Egner A, Geisler C, Von Middendorff C, Bock H, Wenzel D, Medda R . Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys J. 2007; 93(9):3285-90. PMC: 2025649. DOI: 10.1529/biophysj.107.112201. View

20.

Bates M, Huang B, Dempsey G, Zhuang X . Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science. 2007; 317(5845):1749-53. PMC: 2633025. DOI: 10.1126/science.1146598. View