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- W2091654111 abstract "Objective: The emerging field of fluorescence nano-scopy promises to revolutionize biomedical research, and remarkable progress has been made in the measurement of nano-meter distances. Although diffraction of visible light limits the far-field optical resolution to ∼200nm, the center position of spatially resolved fluorescent molecules or nano-particles can be located to much higher precision. Notably, in high resolution microwave and optical spectroscopy there are numerous examples where the line-center is determined with a precision of less than 0.000001 of the line-width. In contrast, the brightest single fluorescent emitters can be detected with a Signal-to-Noise-Ratio of ∼100, limiting the localization precision to 0.01 (∼1.5nm) of the microscope Point-Spread-Function (PSF) width. Moreover the error in co-localizing two or more single emitters is notably worse, remaining greater than 0.03-0.05 (5-10nm) of the PSF width.Results: We achieve two-color single-molecule imaging with 0.5nm absolute localization and registration accuracy as well as demonstrate 0.7nm absolute accuracy in distance measurements between two different color dye-molecules attached at known positions along a surface tethered bio-molecule. The statistical uncertainty in the mean for an ensemble of N∼10 identical single molecule samples is limited only by the total number of collected photons to ∼0.3nm, or ∼0.002 of the width of the optical PSF. We further show how our method can be used to improve the resolution of many sub-wavelength, far-field imaging methods such as those based on co-localization of stochastically excited fluorescent molecules.Conclusion: We demonstrate sub-nanometer resolution in measurements of molecular-scale distances using far-field fluorescence imaging optics, at room temperature and in physiological buffer conditions. The improved resolution will allow deciphering in real-time, at the single molecule level the structure and dynamics of large, multi-subunit biological complexes." @default.
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- W2091654111 date "2009-02-01" @default.
- W2091654111 modified "2023-10-17" @default.
- W2091654111 title "Single-Molecule Fluorescence Imaging with Sub-nanometer Resolution" @default.
- W2091654111 doi "https://doi.org/10.1016/j.bpj.2008.12.2880" @default.
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