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

US8693742B2 · kind B2 · utility

Assignee

• The Regents of the University of Colorado · US

Inventors

• Rafael Piestun · Boulder, US
• Sri Rama Prasanna Pavani · Los Angeles, US
• Michael A. Thompson · Utica, US
• Julie S. Biteen · Menlo Park, US
• William E. Moerner · Stanford, US

Key dates

• Filing date: Dec 17, 2009
• Grant date: Apr 8, 2014
• Expiry date: Aug 20, 2032

Classification

• Technology area (CPC G): Physics
• CPC primary: G06T2207/30024
• WIPO field: Measurement
• WIPO sector: Instruments

Abstract

Embodiments of the present invention can resolve molecules beyond the optical diffraction limit in three dimensions. A double-helix point spread function can be used to in conjunction with a microscope to provide dual-lobed images of a molecule. Based on the rotation of the dual-lobed image, the axial position of the molecule can be estimated or determined. In some embodiments, the angular rotation of the dual-lobed imaged can be determined using a centroid fit calculation or by finding the midpoints of the centers of the two lobes. Regardless of the technique, the correspondence between the rotation and axial position can be utilized. A double-helix point spread function can also be used to determine the lateral positions of molecules and hence their three-dimensional location.