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Optimized localization analysis for single-molecule tracking and super-resolution microscopy.

ABSTRACT: We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

SUBMITTER: Mortensen KI 

PROVIDER: S-EPMC3127582 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Optimized localization analysis for single-molecule tracking and super-resolution microscopy.

Mortensen Kim I KI   Churchman L Stirling LS   Spudich James A JA   Flyvbjerg Henrik H  

Nature methods 20100404 5

We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas max  ...[more]