Sub-diffraction limit image resolution and other imaging techniques

Abstract

The present invention generally relates to sub-diffraction limit 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 entities may be immobilized relative to each other and/or to a common entity. 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 limit 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, a protein, a cell, a tissue, etc. 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 limit image resolution, computer programs and techniques for sub-diffraction limit image resolution, methods for promoting sub-diffraction limit image resolution, methods for producing photoswitchable entities, and the like.

1 . (canceled) 
     
     
         2 . A method of determining spatial information about photoswitchable fluorescent probes in a sample, the method comprising:
 (a) providing a sample labeled with a plurality of photoswitchable fluorescent probes capable of emitting light of at least one wavelength when activated, at least some of the photoswitchable fluorescent probes being separated by a distance of separation less than the at least one wavelength of the emitted light;   (b) exposing the plurality of photoswitchable fluorescent probes to activation light to activate a statistical subset of the plurality of photoswitchable fluorescent probes from a state not capable of emitting light to a state capable of emitting light;   (c) exciting the activated subset with excitation light to cause the activated subset of the plurality of photoswitchable fluorescent probes to emit light;   (d) determining light emitted by the activated subset of the plurality of photoswitchable fluorescent probes;   (e) substantially deactivating the activated subset of the plurality of photoswitchable fluorescent probes;   (f) repeating (b) through (e) one or more times, each time activating a statistically different subset of the photoswitchable fluorescent probes; and   (g) determining the positions of at least some of the photoswitchable fluorescent probes within the sample, to a precision smaller than the wavelength of the emitted light, by using the light emitted by the activated subsets of the photoswitchable fluorescent probes.   
     
     
         3 . The method of  claim 2 , wherein (e) comprises substantially deactivating the activated subset of photoswitchable fluorescent probes by exposing the activated subset of photoswitchable fluorescent probes to deactivation light. 
     
     
         4 . The method of  claim 3 , wherein the excitation light and the deactivation light have substantially the same wavelength. 
     
     
         5 . The method of  claim 2 , wherein (e) comprises substantially deactivating the activated subset of photoswitchable fluorescent probes by waiting for at least a time sufficient to allow the activated subset to substantially spontaneously deactivate. 
     
     
         6 . The method of  claim 2 , further comprising constructing an image using the positions of at least some of the photoswitchable fluorescent probes determined in (g). 
     
     
         7 . The method of  claim 2 , 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. 
     
     
         8 . The method of  claim 2 , 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. 
     
     
         9 . The method of  claim 2 , 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. 
     
     
         10 . The method of  claim 9 , wherein the act of using drift correction comprises using fiduciary markers to determine drift. 
     
     
         11 . The method of  claim 2 , comprising determining the positions of at least some of the photoswitchable fluorescent probes as a function of time. 
     
     
         12 . The method of  claim 2 , wherein at least some of the photoswitchable fluorescent probes comprise a first, light-emitting portion and a second, activation portion that activates the first portion upon exposure to light. 
     
     
         13 . The method of  claim 12 , wherein the first portion is Cy5, Cy5.5, or Cy7, and the second portion is Cy2, Alexa Fluor 488, Cy3, Cy3.5, or Cy5. 
     
     
         14 . The method of  claim 2 , 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. 
     
     
         15 . The method of  claim 2 , wherein substantially all of the photoswitchable fluorescent probes are essentially identical. 
     
     
         16 . The method of  claim 2 , 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.   
     
     
         17 . The method of  claim 2 , wherein at least some of the plurality of photoswitchable fluorescent probes are separated by a distance of less than about 1000 nm. 
     
     
         18 . The method of  claim 2 , 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. 
     
     
         19 . The method of  claim 2 , 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. 
     
     
         20 . The method of  claim 2 , 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 20 nm. 
     
     
         21 . The method of  claim 2 , wherein the plurality of photoswitchable fluorescent probes includes at least a first plurality of photoswitchable fluorescent probes capable of emitting light of at least a first wavelength when activated and a second plurality of photoswitchable fluorescent probes capable of emitting light of at least a second wavelength when activated, wherein the first wavelength and the second wavelength are distinguishable. 
     
     
         22 . The method of  claim 21 , comprising:
 exposing the first plurality of photoswitchable fluorescent probes to first activation light to activate a first statistical subset of the first plurality of photoswitchable fluorescent probes;   exciting the first activated subset with first excitation light to cause the first activated subset of the first plurality of photoswitchable fluorescent probes to emit light;   substantially deactivating the first activated subset of the first plurality of photoswitchable fluorescent probes;   determining light emitted by the first activated subset of the first plurality of photoswitchable fluorescent probes;   exposing the second plurality of photoswitchable fluorescent probes to second activation light to activate a second statistical subset of the second plurality of photoswitchable fluorescent probes;   exciting the second activated subset with second excitation light to cause the second activated subset of the second plurality of photoswitchable fluorescent probes to emit light;   substantially deactivating the second activated subset of the second plurality of photoswitchable fluorescent probes; and   determining light emitted by the second activated subset of the second plurality of photoswitchable fluorescent probes.   
     
     
         23 . The method of  claim 2 , wherein the plurality of photoswitchable fluorescent probes includes at least a first plurality of photoswitchable fluorescent probes able to be activated by light of a first wavelength, and a second plurality of photoswitchable fluorescent probes able to be activated by light of a second wavelength but not the first wavelength. 
     
     
         24 . The method of  claim 23 , comprising:
 exposing the first plurality of photoswitchable fluorescent probes to first activation light to activate a first statistical subset of the first plurality of photoswitchable fluorescent probes;   exciting the first activated subset with first excitation light to cause the first activated subset of the first plurality of photoswitchable fluorescent probes to emit light;   substantially deactivating the first activated subset of the first plurality of photoswitchable fluorescent probes;   determining light emitted by the first activated subset of the first plurality of photoswitchable fluorescent probes;   exposing the second plurality of photoswitchable fluorescent probes to second activation light to activate a second statistical subset of the second plurality of photoswitchable fluorescent probes;   exciting the second activated subset with second excitation light to cause the second activated subset of the second plurality of photoswitchable fluorescent probes to emit light;   substantially deactivating the second activated subset of the second plurality of photoswitchable fluorescent probes; and   determining light emitted by the second activated subset of the second plurality of photoswitchable fluorescent probes.   
     
     
         25 . A method of determining spatial information about photoswitchable fluorescent probes in a sample, the method comprising:
 (a) providing a sample labeled with a plurality of photoswitchable fluorescent probes capable of emitting light of at least one wavelength when activated, at least some of the photoswitchable fluorescent probes being separated by a distance of separation less than the at least one wavelength of the emitted light;   (b) exposing the plurality of photoswitchable fluorescent probes to activation light to activate a statistical subset of the plurality of photoswitchable fluorescent probes from a state not capable of emitting light to a state capable of emitting light;   (c) exciting the activated subset of photoswitchable fluorescent probes with excitation light having an excitation wavelength to cause the activated subset of the plurality of photoswitchable fluorescent probes to emit light;   (d) determining light emitted by the activated subset of the plurality of photoswitchable fluorescent probes;   (e) substantially deactivating the activated subset of the plurality of photoswitchable fluorescent probes using deactivation light having a wavelength substantially identical to the excitation wavelength;   (f) repeating (b) through (e) one or more times, each time activating a statistically different subset of the photoswitchable fluorescent probes; and   (g) determining the positions of at least some of the photoswitchable fluorescent probes within the sample, to a precision smaller than the wavelength of the emitted light, by using the light emitted by the activated subsets of the photoswitchable fluorescent probes.