title: Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function.
abstract: 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.
authors.affiliation: Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA.
authors.forename: Sri Rama Prasanna
authors.initials: SR
authors.lastname: Pavani
authors.forename: Michael A
authors.initials: MA
authors.lastname: Thompson
authors.forename: Julie S
authors.initials: JS
authors.lastname: Biteen
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Lord
authors.forename: Na
authors.initials: N
authors.lastname: Liu
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Twieg
authors.forename: Rafael
authors.initials: R
authors.lastname: Piestun
authors.forename: W E
authors.initials: WE
authors.lastname: Moerner
mesh_terms: D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*