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Subnanometre single-molecule localization, registration and distance measurements


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.


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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 and Affiliations



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.

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Correspondence to Alexandros Pertsinidis or Steven Chu.

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The authors declare no competing financial interests.

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Pertsinidis, A., Zhang, Y. & Chu, S. Subnanometre single-molecule localization, registration and distance measurements. Nature 466, 647–651 (2010).

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