Nanometer-localized multiple single-molecule fluorescence microscopy

被引:238
作者
Qu, XH
Wu, D
Mets, L
Scherer, NF
机构
[1] Univ Chicago, Dept Phys, Chicago, IL 60637 USA
[2] Univ Chicago, Dept Mol Genet & Cell Biol, Chicago, IL 60637 USA
[3] Univ Chicago, Dept Chem, Chicago, IL 60637 USA
[4] Univ Chicago, Inst Biophys Dynam, Chicago, IL 60637 USA
关键词
D O I
10.1073/pnas.0402155101
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.
引用
收藏
页码:11298 / 11303
页数:6
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