Super-resolution orientation estimation and localization of fluorescent dipoles using 3-D steerable filters.
Fluorophores that are fixed during image acquisition produce a diffraction pattern that is characteristic of the orientation of the fluorophore's underlying dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by applying Gaussian-based fitting algorithms to in-focus images of individual fluorophores; when applied to fixed dipoles, this can lead to a bias in the range of 5-20 nm.We introduce a method for the joint estimation of position and orientation of dipoles, based on the representation of a physically realistic image formation model as a 3-D steerable filter. Our approach relies on a single, defocused acquisition. We establish theoretical, localization-based resolution limits on estimation accuracy using Cramér-Rao bounds, and experimentally show that estimation accuracies of at least 5 nm for position and of at least 2 degrees for orientation can be achieved. Patterns generated by applying the image formation model to estimated position/orientation pairs closely match experimental observations.
Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne, Switzerland. francois.aguet@epfl.ch
François
F
Aguet
Stefan
S
Geissbühler
Iwan
I
Märki
Theo
T
Lasser
Michael
M
Unser
D000465:Algorithms; D005456:Fluorescent Dyes; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D012815:Signal Processing, Computer-Assisted
D005456:Fluorescent Dyes