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

• US 8,693,742 B2
• Filed: 12/17/2009
• Issued: 04/08/2014
• 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:
  US12/640,834
• Publication Number:
  US 20100278400A1
• Time in Patent Office: 1,573 Days
• Field of Search: 382/128
• US Class Current: 382/128
• 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 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.

2. The method according to claim 1 wherein the double-helix point spread function is created using an iterative optimization procedure operating in more than one domain.

3. The method according to claim 2, wherein the domains comprise the GL modal plane, the Fourier plane, and the spatial plane.

4. The method according to claim 1 further comprising determining the lateral location relative to an optical axis from the centroid of the point spread.

5. The method according to claim 1, wherein the determining an angular rotation comprises:

finding a center of each of two lobes of the point spread function using a least-squares fit;

determining the midpoint between the two lobe centers; and

determining the angle between the two centers.

6. The method according to claim 1, wherein the determining an angular rotation comprises determining the center of two lobes of the point spread function using one of either a centroid calculation or a Gaussian fit.

7. The method according to claim 1, wherein the distance between the molecule and the focal plane can be determined with an accuracy of about 5-50 nm.

8. The method according to claim 1, wherein the point spread function is a double-helix point spread function.

9. The method according to claim 1, wherein the determining the angular rotation of the point spread determines the rotation of one or more lobes of the point spread.

10. The method according to claim 1 wherein the double-helix point spread function comprises a phase mask.

11. The method according to claim 1 further comprising fluorescent molecules.

12. A fluorescence microscope comprising:

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.

13. The fluorescence microscope according to claim 12, wherein the double-helix point spread function is created using an iterative optimization procedure operating in more than one domain.

14. The fluorescence microscope according to claim 13, wherein the three domains comprise the GL modal plane, the Fourier plane, and the spatial plane.

15. The fluorescence microscope according to claim 12 wherein the double-helix point spread function is disposed within either or both of a phase mask or a spatial light modulator.

16. The fluorescence microscope according to claim 12 further comprising a polarizer disposed between the double-helix point spread function and the imaging device.

17. A method comprising:

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.

18. The method according to claim 17, wherein the determining the center point of the lobes comprises finding the midpoint between the two center points.

19. The method according to claim 17, wherein the determining the center point of the lobes comprises using a centroid fit algorithm.