Backlund, M.P. et al. Proc. Natl. Acad. Sci. USA 109, 19087–19092 (2012).
The quality of super-resolution microscopy based on the localization of individual fluorophores to reconstruct an image with subdiffraction resolution depends on the accuracy of the localization estimates. These estimates are usually based on fitting an isotropic fitting function to each image spot. If the fluorophore dipole is freely rotating, this method provides accurate estimates. But if the dipole is fixed, the resulting anisotropic emission can result in serious localization errors and distorted images. Backlund et al. show how their previously reported double-helix point-spread function can be used to detect and correct mislocalizations caused by nonrotating fluorophores. They can also measure the orientation of the fluorophore. They hope to extend their analysis method to fluorophores that exhibit limited motion, which are probably more commonly observed in biological samples than nonrotating fluorophores.
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Finding and correcting immobile fluorophores. Nat Methods 10, 13 (2013). https://doi.org/10.1038/nmeth.2311
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DOI: https://doi.org/10.1038/nmeth.2311