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Open Access Highly Accessed Question and Answer

Q&A: Single-molecule localization microscopy for biological imaging

Ann L McEvoy, Derek Greenfield, Mark Bates and Jan Liphardt*

BMC Biology 2010, 8:106  doi:10.1186/1741-7007-8-106

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One can localize better than chi-square fitting to a 2D Gaussian

Henrik Flyvbjerg   (2010-09-07 14:31)  Technical University of Denmark email

Q: On Page 6 you write that "Once a target fluorophore is found, the signal is fitted to a 2D Gaussian distribution (or the centroid of the signal is determined)."

Cannot one do better than using a Gaussian?
If not, how should the fitting be done?
With ordinary least squares, weighted least squares, or maximum likelihood fitting?

A: Yes, for fluorophores with fixed orientation, a 2D Gaussian fit is not optimal at all. Better expressions for the PSF are available. For fluorescent beads a 2D Gaussian is essentially optimal. One should, however, fit with maximum likelihood, not least squares, in order to localize optimally.

See "Optimized localization-analysis for single-molecule tracking and super-resolution microscopy" by
Kim I. Mortensen, L. Stirling Churchman, James A. Spudich, and Henrik Flyvbjerg
Nature Methods 7, 377-381 (2010); doi:10.1038/nmeth.1447

Competing interests

None declared

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