Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 9, Pages 2995-2999Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0900245106
Keywords
microscopy; photoactivation; superresolution; computational imaging; PSF engineering
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Funding
- National Institute of General Medical Sciences [R01GM085437]
- National Science Foundation Award [NIRT-0304650]
- Technology Transfer Office of the University of Colorado
- CDM Optics PhD fellowship
- National Science Foundation Graduate Fellowship
- Stanford Graduate Fellowship
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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 mu m) 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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