Sub-diffraction image resolution and other imaging techniques
The present invention generally relates to sub-diffraction image resolution and other imaging techniques. 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. For example, the entities may be separated by a distance of less than about 1000 nm, or less than about 300 nm for visible light. In one set of embodiments, the entities may be selectively activatable, i.e., one entity can be activated to produce light, without activating other entities. A first entity may be activated and determined (e.g., by determining light emitted by the entity), then a second entity may be activated and determined. The emitted light may be used to determine the positions of the first and second entities, for example, using Gaussian fitting or other mathematical techniques, and in some cases, with sub-diffraction resolution. The methods may thus be used, for example, to determine the locations of two or more entities immobilized relative to a common entity, for example, a surface, or a biological entity such as DNA or a protein. The entities may also be determined with respect to time, for example, to determine a time-varying reaction. Other aspects of the invention relate to systems for sub-diffraction image resolution, computer programs and techniques for sub-diffraction image resolution, methods for promoting sub-diffraction image resolution, methods for producing photoswitchable entities, and the like.
1. A method of imaging a sample comprising photoswitchable fluorescent probes, the method comprising:
(a) providing a sample labeled with a plurality of photoswitchable fluorescent probes, at least some of the photoswitchable fluorescent probes being separated by a distance of less than about 1000 nm, at least some of the photoswitchable fluorescent probes being in a state not capable of emitting light at a first wavelength;
(b) exposing the plurality of photoswitchable fluorescent probes to substantially identical activation light to activate a statistical subset of the photoswitchable fluorescent probes from a state not capable of emitting light at the first wavelength to a state capable of emitting light at the first wavelength;
(c) exciting the activated subset of photoswitchable fluorescent probes with excitation light to cause the activated subset to emit light at the first wavelength;
(d) determining light emitted at the first wavelength by the activated subset of photoswitchable fluorescent probes;
(e) substantially deactivating the activated subset of photoswitchable fluorescent probes;
(f) repeating (b) through (e) one or more times, each time activating a statistically different subset of the plurality of photoswitchable fluorescent probes; and
(g) determining the positions of at least some of the photoswitchable fluorescent probes within the sample by using the light emitted by the activated subsets of the photoswitchable fluorescent probes.
2. The method of claim 1 , wherein the act of determining light emitted by the activated subsets of photoswitchable probes comprises acquiring an image of the light emitted by the activated subsets of the photoswitchable fluorescent probes.
3. The method of claim 1 , wherein the act of determining the positions of at least some of the photoswitchable fluorescent probes comprises using Gaussian fitting of the light emitted by the activated subsets of the photoswitchable fluorescent probes.
4. The method of claim 1 , wherein the positions of at least some of the photoswitchable fluorescent probes are determined to a precision that is smaller than the first wavelength.
5. The method of claim 4 , wherein the precision defined by the full width at half maximum (FWHM) is smaller than about 20 nm.
6. The method of claim 1 , wherein the act of determining the positions of at least some of the photoswitchable fluorescent probes comprises using drift correction to determine the positions of at least some of the photoswitchable fluorescent probes.
7. The method of claim 6 , wherein the act of using drift correction comprises using fiduciary markers to determine drift.
8. The method of claim 1 , comprising determining the positions of at least some of the photoswitchable fluorescent probes as a function of time.
9. The method of claim 1 , wherein at least some of the photoswitchable fluorescent probes comprises a first, light-emitting portion and a second, activation portion that activates the first portion upon exposure to light.
10. The method of claim 9 , wherein the first portion is Cy5, Cy5.5, or Cy7 and the second portion is Cy2, Alexa Fluor 488, Cy3, Cy3.5, or Cy5.
11. The method of claim 1 , wherein the activation light of (b) has an intensity sufficient to activate, on average, a statistical subset but not all of the photoswitchable fluorescent probes that the activation light is incident thereto.
12. The method of claim 1 , wherein (e) comprises substantially deactivating the activated subset of photoswitchable fluorescent probes by exposing at least the activated subset of photoswitchable fluorescent probes to deactivation light.
13. The method of claim 1 , wherein substantially all of the photoswitchable fluorescent probes are essentially identical.
14. The method of claim 1 , wherein (a) comprises:
providing a sample labeled with a plurality of photoswitchable fluorescent probes; and
deactivating at least some of the photoswitchable fluorescent probes by exposing the photoswitchable fluorescent probes to deactivation light such that at least some of the photoswitchable fluorescent probes are placed in a state not capable of emitting light at a first wavelength.
15. The method of claim 1 , wherein (g) comprises determining the positions of at least some of the photoswitchable fluorescent probes within the sample to a precision of less than about 300 nm.
16. The method of claim 1 , wherein (g) comprises determining the positions of at least some of the photoswitchable fluorescent probes within the sample to a precision of less than about 100 nm.
17. The method of claim 1 , wherein (g) comprises determining the positions of at least some of the photoswitchable fluorescent probes within the sample to a precision of less than about 40 nm.
18. The method of claim 1 , wherein (g) comprises determining the positions of at least some of the photo switchable fluorescent probes within the sample to a precision of less than about 20 nm.