Three-Dimensional, Single-Molecule Fluorescence Imaging Beyond the Diffraction Limit by using a Double-Helix Point Spread Function

Sri Rama Prasanna Pavani, Michael A. Thompson, Julie S. Biteen, Samuel J. Lord, Na Liu, Robert J. Twieg, Rafael Piestun, W. E. Moerner

PNAS March 3, 2009 106 (9) 2995-2999;


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 μm) 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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