THREE-DIMENSIONAL SINGLE-MOLECULE FLUORESCENCE IMAGING BEYOND THE DIFFRACTION LIMIT USING A DOUBLE-HELIX POINT SPREAD FUNCTION
First Claim
1. A method comprising:
- illuminating a molecule near a focal plane of an imaging system;
directing light from the molecules toward an optical element implementing comprising a double-helix point spread function;
imaging a point spread produced by the system'"'"'s point spread function as a response to the molecule'"'"'s emission;
determining an angular rotation of the point spread relative to a baseline rotation; and
determining the distance between the molecule and the imaging plane from the angular rotation.
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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.
60 Citations
20 Claims
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1. A method comprising:
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illuminating a molecule near a focal plane of an imaging system; directing light from the molecules toward an optical element implementing comprising a double-helix point spread function; imaging a point spread produced by the system'"'"'s point spread function as a response to the molecule'"'"'s emission; determining an angular rotation of the point spread relative to a baseline rotation; and determining the distance between the molecule and the imaging plane from the angular rotation. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A fluorescence microscope comprising:
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an illumination system configured to illuminate a fluorescing molecule at an illumination wavelength; an optical system configured to image the fluorescing molecule at an imaging wavelength, wherein the imaging wavelength is different than the illumination wavelength; a double-helix point spread function disposed within the optical system, the double-helix point spread function configured to produce a double-helix point spread that rotates as a function of the fluorescing material'"'"'s distance from an imaging plane; and an imager configured to produce an image of the double helix point spread. - View Dependent Claims (13, 14, 15, 16, 17)
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18. A method comprising:
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receiving a dual lobe image of a molecule through a double helix point spread function; determining a point of maximum intensity of each lobe; determining the angular rotation of the lobes using the center point; and determining the position of the molecule from the angular rotation of the lobes. - View Dependent Claims (19, 20)
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Specification