Three-dimensional single-molecule fluorescence imaging beyond the diffraction limit using a double-helix point spread function
First Claim
1. A method comprising:
- illuminating with an illumination system of an imaging system a molecule within a sample near a focal plane of the imaging system;
directing light from the molecule toward an optical element comprising a double-helix point spread function;
imaging a point spread produced by the double-helix point spread function as a response to the molecule'"'"'s emission;
determining, with a processor, an angular rotation of the point spread relative to a baseline rotation;
determining, with a processor, the distance between the molecule and the focal plane from the angular rotation; and
generating an output that when displayed specifies an estimated position of the molecule within the sample.
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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.
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Citations
19 Claims
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1. A method comprising:
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illuminating with an illumination system of an imaging system a molecule within a sample near a focal plane of the imaging system; directing light from the molecule toward an optical element comprising a double-helix point spread function; imaging a point spread produced by the double-helix point spread function as a response to the molecule'"'"'s emission; determining, with a processor, an angular rotation of the point spread relative to a baseline rotation; determining, with a processor, the distance between the molecule and the focal plane from the angular rotation; and generating an output that when displayed specifies an estimated position of the molecule within the sample. - 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 a focal plane; an imager configured to produce an image of the double helix point spread; and a processor configured to; determine an angular rotation of a double-helix point spread in the image, determine a distance between the fluorescing material and an focal plane using the angular rotation; and output the distance between the fluorescing material and the focal plane. - View Dependent Claims (13, 14, 15, 16)
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17. A method comprising:
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receiving from an imager of an optical system a dual lobe image of a molecule through a double helix point spread function disposed within the optical system; determining a point of maximum intensity of each lobe; determining, with a processor, the angular rotation of the lobes using the center point; determining, with a processor, the position of the molecule from the angular rotation of the lobes; and outputting at least one of an x-coordinate, a y-coordinate, and a z-coordinate that specifies the position of the molecule. - View Dependent Claims (18, 19)
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Specification