Sub-diffraction 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 . 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 having a sufficiently weak intensity such that the incident light activates 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 statistical subset of photoswitchable fluorescent probes with excitation light having an excitation wavelength to cause the activated statistical subset of photoswitchable fluorescent probes to emit light at the emission wavelength, and to subsequently deactivate upon continued exposure to the excitation light;   (d) determining the light emitted by the statistical subset of photoswitchable fluorescent probes prior to deactivation of the statistical subset of photoswitchable fluorescent probes;   (e) repeating (b) through (d) one or more times, each time causing a statistically different subset of the photoswitchable fluorescent probes to emit light; and   (f) 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 statistical subsets of the photoswitchable fluorescent probes.   
     
     
         2 . The method of  claim 1 , further comprising constructing an image using the positions of at least some of the fluorescent probes determined in (f). 
     
     
         3 . The method of  claim 2 , wherein the image comprises at least three colors based on the wavelengths of the emitted light. 
     
     
         4 . The method of  claim 1 , wherein the act of determining the positions of at least some of the fluorescent probes comprises using Gaussian fitting of the light emitted by the statistical subset of the plurality of fluorescent probes. 
     
     
         5 . The method of  claim 1 , comprising determining the positions of at least some of the fluorescent probes as a function of time. 
     
     
         6 . The method of  claim 1 , further comprising constructing an image using the positions at least some of the fluorescent probes in no more than 30 min of imaging time. 
     
     
         7 . The method of  claim 1 , wherein the act of determining the light emitted by the statistical subset of the plurality of fluorescent probes comprises acquiring an image of the light emitted by the statistical subset of the plurality of fluorescent probes. 
     
     
         8 . The method of  claim 1 , wherein the act of determining the positions of at least some of the fluorescent probes comprises using drift correction to determine the positions of at least some of the fluorescent probes. 
     
     
         9 . The method of  claim 8 , wherein the act of using drift correction comprises using fiduciary markers to determine drift. 
     
     
         10 . The method of  claim 1 , wherein the precision is smaller than about 700 nm. 
     
     
         11 . The method of  claim 1 , wherein the precision is smaller than about 100 nm. 
     
     
         12 . The method of  claim 1 , wherein (d) comprises acquiring the emitted light using a camera 
     
     
         13 . The method of  claim 12 , wherein the timing of the activating light and the frames of the camera are synchronized. 
     
     
         14 . The method of  claim 1 , wherein at least some of the fluorescent probes comprise Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DiD, DiR, YOYO-3, YO-PRO-3, TOT-3, and/or TO-PRO-3. 
     
     
         15 . The method of  claim 1 , wherein substantially all of the fluorescent probes in the sample are essentially identical. 
     
     
         16 . The method of  claim 1 , wherein at least some of the plurality of fluorescent probes are separated by a distance of less than about 1000 nm. 
     
     
         17 . The method of  claim 1 , wherein at least some of the fluorescent probes comprise Cy5-Alexa Fluor 405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3, Cy5-Cy3.5, Cy5.5-Alexa Fluor 405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2, Cy5.5-Cy3, Cy5.5-Cy3.5, Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488, Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5, Alexa Fluor 647-Alexa Fluor 405, Alexa Fluor 647-Alexa Fluor 488, Alexa Fluor 647-Cy2, Alexa Fluor 647-Cy3, and/or Alexa Fluor 647-Cy3.5.