Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy.
We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.
German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. shell@gwdg.de
Johann
J
Engelhardt
Jan
J
Keller
Patrick
P
Hoyer
Matthias
M
Reuss
Thorsten
T
Staudt
Stefan W
SW
Hell
D005456:Fluorescent Dyes / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012680:Sensitivity and Specificity; D013269:Stochastic Processes
D005456:Fluorescent Dyes