Sub-diffraction limit image resolution in three dimensions
The present invention generally relates to sub-diffraction limit image resolution and other imaging techniques, including imaging in three dimensions. In one aspect, the invention is directed to determining and/or imaging light from two or more entities separated by a distance less than the diffraction limit of the incident light. In some cases, the position of the entities can be determined in all three spatial dimensions (i.e., in the x, y, and z directions), and in certain cases, the positions in all three dimensions can be determined to an accuracy of less than about 1000 nm. In some cases, the z positions may be determined using one of a variety of techniques that uses intensity information or focal information (e.g., a lack of focus) to determine the z position. Non-limiting examples of such techniques include astigmatism imaging, off-focus imaging, or multi-focal-plane imaging.
1. A method, comprising:
providing a plurality of entities able to emit light;
activating a fraction of the plurality of entities to emit light;
determining the emitted light;
deactivating the activated fraction of the plurality of entities; and
repeating the acts of activating and deactivating the plurality of entities to determine x, y, and z positions of the plurality of entities, wherein the x, y, and z positions of the plurality of entities are 3-D spatial coordinates and are each determined to a precision better than the wavelength of the light emitted by some of the plurality of entities, and wherein the z positions of the plurality of entities are determined using astigmatism imaging.
2. The method of claim 1 , wherein at least some of the plurality of entities are separated by a distance of separation less than the wavelength of the emitted light.
3. The method of claim 1 , further comprising constructing an image of the plurality of entities using their respective x, y, and z positions.
4. The method of claim 1 , comprising passing light emitted by the fraction of the plurality of entities through a lens that is non-circularly symmetric with respect to the direction light emitted by the some of the plurality of entities passes through the lens.
5. The method of claim 1 , further comprising passing light emitted by the fraction of the plurality of entities through a cylindrical lens.
6. The method of claim 1 , further comprising collecting the light emitted by the fraction of the plurality of entities with a detector, wherein the light emitted by the some of the fraction of the plurality of entities is not focused at the detector.
7. The method of claim 6 , further comprising collecting the light emitted by the fraction of the plurality of entities by a plurality of detectors, wherein some of the fraction of the plurality of entities are not in a focal region of the plurality of detectors.
8. The method of claim 6 , comprising passing the light emitted by the fraction of the plurality of entities through a lens that defines a focal region that does not contain at least a portion of the fraction of the plurality of entities.
9. The method of claim 1 , further comprising collecting the light emitted by the first entity with a detector, wherein the first entity is not in a conjugate plane of the detector.
10. The method of claim 1 , further comprising collecting the light emitted by the fraction of the plurality of entities by a plurality of detectors, wherein at one or more of the plurality of detectors, the light emitted by at least a portion of the fraction of the plurality of entities is not focused.
11. The method of claim 1 , wherein at least some of the plurality of entities emit light at different wavelengths.
12. The method of claim 1 , wherein the plurality of entities each emit light at substantially the same wavelength.
13. The method of claim 1 , wherein at least some of the plurality of entities are activated by light at different wavelengths.
14. The method of claim 1 , wherein the plurality of entities are each activated by light at substantially the same wavelength.
15. The method of claim 1 , wherein at least some of the plurality of entities are photoactivatable or photoswitchable probes.
16. The method of claim 1 , wherein the light emitted by at least some of the plurality of entities has a wavelength of between about 400 nm and about 1000 nm.
17. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 1000 nm.
18. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 300 nm.
19. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 100 nm.
20. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 50 nm.
21. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 20 nm.
22. The method of claim 1 , wherein the positions of the plurality of entities are determined to a precision better than about 10 nm.
23. The method of claim 1 , comprising determining the positions of the plurality of entities at a first point of time, and determining the positions of the plurality of entities at a second point of time.
24. The method of claim 1 , wherein the acts are performed in the order recited.