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Subnanometre single-molecule localization, registration and distance measurements

Abstract

Remarkable progress in optical microscopy has been made in the measurement of nanometre distances. If diffraction blurs the image of a point object into an Airy disk with a root-mean-squared (r.m.s.) size of s =  0.44λ/2NA (90 nm for light with a wavelength of λ = 600 nm and an objective lens with a numerical aperture of NA = 1.49), limiting the resolution of the far-field microscope in use to d = 2.4s ≈ 200 nm, additional knowledge about the specimen can be used to great advantage. For example, if the source is known to be two spatially resolved fluorescent molecules, the distance between them is given by the separation of the centres of the two fluorescence images1. In high-resolution microwave and optical spectroscopy, there are numerous examples where the line centre is determined with a precision of less than 10−6 of the linewidth. In contrast, in biological applications the brightest single fluorescent emitters can be detected with a signal-to-noise ratio of 100, limiting the centroid localization precision to sloc ≥ 1% (≥1 nm) of the r.m.s. size, s, of the microscope point spread function (PSF)2. Moreover, the error in co-localizing two or more single emitters is notably worse, remaining greater than 5–10% (5–10 nm) of the PSF size3,4,5,6,7,8. Here we report a distance resolution of sreg = 0.50 nm (1σ) and an absolute accuracy of sdistance = 0.77 nm (1σ) in a measurement of the separation between differently coloured fluorescent molecules using conventional far-field fluorescence imaging in physiological buffer conditions. The statistical uncertainty in the mean for an ensemble of identical single-molecule samples is limited only by the total number of collected photons, to sloc ≈ 0.3 nm, which is 3 × 10−3 times the size of the optical PSF. Our method may also be used to improve the resolution of many subwavelength, far-field imaging methods such as those based on co-localization of molecules that are stochastically switched on in space6,7,8. The improved resolution will allow the structure of large, multisubunit biological complexes in biologically relevant environments to be deciphered at the single-molecule level.

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Figure 1: Active feedback control.
Figure 2: Mapping function calibrations.
Figure 3: Distance measurements on optically manipulated molecules.
Figure 4: Measurements of molecular-scale structures using same-colour probes.

References

  1. Bobroff, N. Position measurement with a resolution and noise-limited instrument. Rev. Sci. Instrum. 57, 1152–1157 (1986)

    ADS  Article  Google Scholar 

  2. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003)

    ADS  CAS  Article  Google Scholar 

  3. Gordon, M. P., Ha, T. & Selvin, P. R. Single-molecule high-resolution imaging with photobleaching. Proc. Natl Acad. Sci. USA 101, 6462–6465 (2004)

    ADS  CAS  Article  Google Scholar 

  4. Qu, X. et al. Nanometer-localized multiple single-molecule fluorescence microscopy. Proc. Natl Acad. Sci. USA 101, 11298–11303 (2004)

    ADS  CAS  Article  Google Scholar 

  5. Ram, S., Ward, E. S. & Ober, R. J. Beyond Rayleigh’s criterion: a resolution measure with application to single-molecule microscopy. Proc. Natl Acad. Sci. USA 103, 4457–4462 (2006)

    ADS  CAS  Article  Google Scholar 

  6. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006)

    ADS  CAS  Article  Google Scholar 

  7. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–796 (2006)

    CAS  Article  Google Scholar 

  8. Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006)

    ADS  CAS  Article  Google Scholar 

  9. Burns, D. H. et al. Strategies for attaining superresolution using spectroscopic data as constraints. Appl. Opt. 24, 154–161 (1985)

    ADS  CAS  Article  Google Scholar 

  10. Bornfleth, H. et al. High-precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy. J. Microsc. 189, 118–136 (1998)

    Article  Google Scholar 

  11. Betzig, E. Proposed method for molecular optical imaging. Opt. Lett. 20, 237–239 (1995)

    ADS  CAS  Article  Google Scholar 

  12. van Oijen, A. M. et al. Far-field fluorescence microscopy beyond the diffraction limit. J. Opt. Soc. Am. A 16, 909–915 (1999)

    ADS  CAS  Article  Google Scholar 

  13. Lacoste, T. D. et al. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc. Natl Acad. Sci. USA 97, 9461–9466 (2000)

    ADS  CAS  Article  Google Scholar 

  14. Antelman, J. et al. Nanometer distance measurements between multicolor quantum dots. Nano Lett. 9, 2199–2205 (2009)

    ADS  CAS  Article  Google Scholar 

  15. Koyama-Honda, I. et al. Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. Biophys. J. 88, 2126–2136 (2005)

    CAS  Article  Google Scholar 

  16. Churchman, L. S. et al. Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc. Natl Acad. Sci. USA 102, 1419–1423 (2005)

    ADS  CAS  Article  Google Scholar 

  17. Heilemann, M. et al. High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy. Anal. Chem. 74, 3511–3517 (2002)

    CAS  Article  Google Scholar 

  18. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)

    ADS  CAS  Article  Google Scholar 

  19. Klar, T. A. et al. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000)

    ADS  CAS  Article  Google Scholar 

  20. Rittweger, E. et al. STED microscopy reveals crystal colour centres with nanometric resolution. Nature Photon. 3, 144–147 (2009)

    ADS  CAS  Article  Google Scholar 

  21. Lauer, T. R. The photometry of undersampled point-spread functions. Publ. Astron. Soc. Pacif. 111, 1434–1443 (1999)

    ADS  Article  Google Scholar 

  22. Boggon, T. J. et al. C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296, 1308–1313 (2002)

    ADS  CAS  Article  Google Scholar 

  23. Pertz, O. et al. A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation. EMBO J. 18, 1738–1747 (1999)

    CAS  Article  Google Scholar 

  24. He, W., Cowin, P. & Stokes, D. L. Untangling desmosomal knots with electron tomography. Science 302, 109–113 (2003)

    ADS  CAS  Article  Google Scholar 

  25. Chappuis-Flament, S. et al. Multiple cadherin extracellular repeats mediate homophilic binding and adhesion. J. Cell Biol. 154, 231–243 (2001)

    CAS  Article  Google Scholar 

  26. Perret, E. et al. Trans-bonded pairs of E-cadherin exhibit a remarkable hierarchy of mechanical strengths. Proc. Natl Acad. Sci. USA 101, 16472–16477 (2004)

    ADS  CAS  Article  Google Scholar 

  27. Zhang, Y. et al. Resolving cadherin interactions and binding cooperativity at the single-molecule level. Proc. Natl Acad. Sci. USA 106, 109–114 (2009)

    ADS  CAS  Article  Google Scholar 

  28. Pokutta, S. et al. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding. Eur. J. Biochem. 223, 1019–1026 (1994)

    CAS  Article  Google Scholar 

  29. Koch, A. W. et al. Calcium binding and homoassociation of E-cadherin domains. Biochemistry 36, 7697–7705 (1997)

    CAS  Article  Google Scholar 

  30. Nagar, B. et al. Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 380, 360–364 (1996)

    ADS  CAS  Article  Google Scholar 

  31. Sivasankar, S. et al. Characterizing the initial encounter complex in cadherin adhesion. Structure 17, 1075–1081 (2009)

    CAS  Article  Google Scholar 

  32. Kingshott, P., Thissen, H. & Griesser, H. J. Effects of cloud-point grafting, chain length, and density of PEG layers on competitive adsorption of ocular proteins. Biomaterials 23, 2043–2056 (2002)

    CAS  Article  Google Scholar 

  33. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006)

    CAS  Article  Google Scholar 

  34. Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Edn Engl. 47, 5465–5469 (2008)

    CAS  Article  Google Scholar 

  35. Cheezum, M. K., Walker, W. F. & Guilford, W. H. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81, 2378–2388 (2001)

    CAS  Article  Google Scholar 

  36. Speidel, M., Jonáš, A. & Florin, E. Three-dimensional tracking of fluorescent nanoparticles with subnanometer precision by use of off-focus imaging. Opt. Lett. 28, 69–71 (2003)

    ADS  CAS  Article  Google Scholar 

  37. Lang, M. J. et al. Simultaneous, coincident optical trapping and single-molecule fluorescence. Nature Methods 1, 133–139 (2004)

    CAS  Article  Google Scholar 

  38. van Dijk, M. A. et al. Combining optical trapping and single-molecule fluorescence spectroscopy: enhanced photobleaching of fluorophores. J. Phys. Chem. B 108, 6479–6484 (2004)

    CAS  Article  Google Scholar 

  39. Brau, R. R. et al. Interlaced optical force-fluorescence measurements for single molecule biophysics. Biophys. J. 91, 1069–1077 (2006)

    ADS  CAS  Article  Google Scholar 

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Acknowledgements

A.P. wishes to thank S. R. Park for assistance with sample preparation and N. Gemelke, E. Sarajlic and H. Mueller for advice on electronics construction. Y.Z. is grateful to J. Nelson and A. Brunger for generously making laboratory facilities available. We thank A. Brunger for critical reading of the manuscript. This work was supported by the National Institutes of Health, the National Science Foundation, the National Aeronautics and Space Administration and the Defense Advanced Research Projects Agency.

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Authors

Contributions

A.P. and S.C. conceived the idea. A.P. designed the experiments. A.P. developed and constructed the measurement apparatus. A.P. and Y.Z. wrote software to analyse the data. A.P. and Y.Z. prepared the reagents and samples. A.P. and Y.Z. developed the mapping function calibrations. A.P. performed the single-molecule measurements and data analysis. S.C. supervised the work and provided guidance. A.P. and S.C. wrote the manuscript.

Corresponding authors

Correspondence to Alexandros Pertsinidis or Steven Chu.

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The authors declare no competing financial interests.

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This file contains Supplementary Methods, a Supplementary Discussion, Supplementary Tables 1-3, References and Supplementary Figures 1-19 with legends. (PDF 833 kb)

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Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010). https://doi.org/10.1038/nature09163

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