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

Kim Mortensen; L. S. Churchman; J. A. Spudich; Henrik Flyvbjerg

doi:10.1038/NMETH.1447

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

Kim Mortensen
, L. S. Churchman
, J. A. Spudich
, Henrik Flyvbjerg

Research output: Contribution to journal › Journal article › Research › peer-review

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.

Original language	English
Journal	Nature Methods
Volume	7
Issue number	5
Pages (from-to)	377-U59
ISSN	1548-7091
DOIs	https://doi.org/10.1038/NMETH.1447
Publication status	Published - 2010

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title = "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.",

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year = "2010",

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T1 - Optimized localization analysis for single-molecule tracking and super-resolution microscopy

AU - Mortensen, Kim

AU - Churchman, L. S.

AU - Spudich, J. A.

AU - Flyvbjerg, Henrik

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

U2 - 10.1038/NMETH.1447

DO - 10.1038/NMETH.1447

M3 - Journal article

SN - 1548-7091

VL - 7

SP - 377-U59

JO - Nature Methods

JF - Nature Methods

IS - 5

ER -

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

Abstract

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