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Super-Resolution Orientation Estimation and Localization of Fluorescent Dipoles Using 3-D Steerable Filters

F. Aguet, S. Geissbühler, I. Märki, T. Lasser, M. Unser

Optics Express, vol. 17, no. 8, pp. 6829-6848, April 9, 2009.

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.

AUTHOR="Aguet, F. and Geissb{\"{u}}hler, S. and M{\"{a}}rki, I. and
        Lasser, T. and Unser, M.",
TITLE="Super-Resolution Orientation Estimation and Localization of
        Fluorescent Dipoles Using \mbox{3-D} Steerable Filters",
JOURNAL="Optics Express",
month="April 9,",

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