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Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):2995-9. doi: 10.1073/pnas.0900245106. Epub 2009 Feb 11.

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function.

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Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA.


We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

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