Optimized localization analysis for single-molecule tracking and super-resolution microscopy.
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.
Mortensen
Kim I
KI
Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.
Churchman
L Stirling
LS
Spudich
James A
JA
Flyvbjerg
Henrik
H
D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D016013:Likelihood Functions; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology; D017785:Photons