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

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

Proceedings of the National Academy of Sciences - PNAS, 2009-03, Vol.106 (9), p.2995-2999 [Peer Reviewed Journal]

United States: National Academy of Sciences

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Title:
Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function
Author: Pavani, Sri Rama Prasanna ; Thompson, Michael A ; Biteen, Julie S ; Lord, Samuel J ; Liu, Na ; Twieg, Robert J ; Piestun, Rafael ; Moerner, W.E
Subjects: Biochemistry ; Calibration ; Diffraction ; Fluorescence ; Geodetic position ; Imaging ; Imaging, Three-Dimensional - instrumentation ; Imaging, Three-Dimensional - methods ; Laser beams ; Microscopes ; Microscopy ; Microscopy, Fluorescence - instrumentation ; Microscopy, Fluorescence - methods ; Molecules ; Photons ; Physical Sciences ; Polymers ; Three dimensional imaging ; Wave diffraction
Is Part Of: Proceedings of the National Academy of Sciences - PNAS, 2009-03, Vol.106 (9), p.2995-2999
Notes: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
1S.R.P.P. and M.A.T. contributed equally to this work.
Author contributions: S.R.P.P., M.A.T., R.P., and W.E.M. designed research; S.R.P.P., M.A.T., J.S.B., R.P., and W.E.M. performed research; S.R.P.P., M.A.T., S.J.L., N.L., and R.J.T. contributed new reagents/analytic tools; S.R.P.P. and M.A.T. analyzed data; and S.R.P.P., M.A.T., J.S.B., S.J.L., R.J.T., R.P., and W.E.M. wrote the paper.
Contributed by W. E. Moerner, January 9, 2009
Description: 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.
Publisher: United States: National Academy of Sciences
Language: English

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

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

United States: National Academy of Sciences

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