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

• US 9,881,355 B2
• Filed: 02/12/2014
• Issued: 01/30/2018
• Est. Priority Date: 12/17/2008
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.), The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Piestun, Rafael, Pavani, Sri Rama Prasanna, Thompson, Michael A., Biteen, Julie S., Moerner, William E.
• Primary Examiner(s): NGUYEN, TRAN N
• Application Number:
  US14/179,546
• Publication Number:
  US 20140226881A1
• Time in Patent Office: 1,448 Days
• Field of Search: 382128
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 3/4053 based on super-resolution, i.e. the output image resolution being higher than the sensor resolution
• CPC Class Codes: G06T 7/12 Edge-based segmentation

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.

Claims

1. A microscope comprising:

an illumination system configured to illuminate at least one particle; and

an optical system configured to image light from the at least one particle, the optical system comprising;

an imager;

an optical element configured to modulate light from the illumination system so that the light includes two intensity spots separated by a first distance along a first axis and separated by a second distance along a second axis, wherein the second distance depends on the axial position of the particle, and wherein the first axis and the second axis are orthogonal; and

one or more lenses configured to direct light from the illumination system and the optical element to the at least one particle, and direct light from the at least one particle to the imager.

2. The microscope according to claim 1, wherein the optical element comprises a phase mask.

3. The microscope according to claim 1, wherein the optical element generates a point spread function.

4. The microscope according to claim 1, wherein the optical element generates a double-helix point spread function.

5. The microscope according to claim 1, wherein the optical element comprises a spatial light modulator.

6. The microscope according to claim 1, wherein the optical element comprises a diffractive optical element.

7. The microscope according to claim 1, wherein the optical element is disposed within a Fourier plane of the optical system.

8. The microscope according to claim 1, further comprising a processor configured to determine an axial distance to the at least one particle based on the second distance.

9. The microscope according to claim 1, further comprising a processor configured to determine an axial distance between the at least one particle and an imaging plane of the microscope based on the second distance.

10. The microscope according to claim 1, further comprising a processor configured to determine an axial distance between the at least one particle and an imaging plane of the microscope based on the position of the two intensity spots.

11. A method comprising:

directing light from at least one particle toward an optical system that generates a point spread function; and

imaging light through the optical system to produce an image of the point spread function that includes two intensity spots separated by a first distance along a first axis and separated by a second distance along a second axis, wherein either or both the first distance or the second distance varies based on the axial position of the at least one particle, and wherein the first axis and the second axis are orthogonal.

12. The method according to claim 11, further comprising illuminating at least the one particle.

13. The method according to claim 11, wherein the optical system comprises a phase mask.

14. The method according to claim 11, wherein the point spread function is a double-helix point spread function.

15. The method according to claim 11, wherein the optical system comprises a spatial light modulator.

16. The method according to claim 11, wherein the optical system comprises a diffractive optical element.

17. The method according to claim 11, wherein the optical system is disposed within a Fourier plane of the optical system.

18. The method according to claim 11, further determining an axial distance to the at least one particle based on the second distance.

19. The method according to claim 11, further comprising a processor configured to determine an axial distance between the at least one particle and an imaging plane of the microscope based on the second distance.

20. A microscope comprising:

an illumination system configured to illuminate at least one particle;

an optical system configured to image light from the at least one particle, the optical system comprising;

an imager;

an optical element configured to modulate light from the illumination system so that the light includes two intensity spots, wherein a position of the two intensity spots depends on the axial distance between the at least one particle and an imaging plane of the optical system; and

one or more lenses configured to direct light from the optical element and the illumination system to the at least one particle and direct light from the at least one particle to the imager.

21. The microscope according to claim 20, wherein the optical element comprises a spatial light modulator.