Skip to main content

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

Back to article

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

    Henrik Flyvbjerg, Technical University of Denmark

    7 September 2010

    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