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 - 227 . (canceled) 
     
     
         228 . A photoswitchable imaging composition, comprising:
 a light-emitting molecular entity, capable of being switched between a first state able to emit light at a first, emission wavelength upon excitation and a second state that does not substantially emit light at the first wavelength upon excitation,   wherein the light-emitting entity comprises a first portion that is capable of emitting light at the first wavelength upon excitation when the light-emitting entity is in the first state, and a second portion that activates the first portion upon exposure to an external stimulus, thereby placing the light-emitting entity in the first state, and   wherein the first portion and the second portion are each bonded to a common protein and are not bonded directly to each other.   
     
     
         229 . The imaging composition of  claim 228 , wherein the second portion that activates the first portion upon exposure to light having an activation wavelength. 
     
     
         230 . The imaging composition of  claim 228 , wherein the first portion, when isolated from the second portion, is fluorescent. 
     
     
         231 . The imaging composition of  claim 228 , wherein the second portion, when isolated from the first portion, is fluorescent. 
     
     
         232 . The imaging composition of  claim 228 , wherein the first portion is 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, TOTO-3, or TO-PRO-3. 
     
     
         233 . The imaging composition of  claim 228 , wherein the second portion is Alexa Fluor 405, Cy2, Alexa Fluor 488, Cy3, Cy3B, or Cy3.5. 
     
     
         234 . The imaging composition of  claim 228 , wherein the first portion and the second portion are separated by a distance of no more than about 50 nm. 
     
     
         235 . The imaging composition of  claim 228 , wherein the common biological protein is an antibody. 
     
     
         236 . The imaging composition of  claim 228 , wherein the first portion comprises two heteroaromatic moieties each comprising a quaternized nitrogen, joined by a polymethine chain. 
     
     
         237 . The imaging composition of  claim 236 , wherein the polymethine chain comprises at least 5 carbon atoms. 
     
     
         238 . The imaging composition of  claim 228 , wherein the first wavelength of emitted light is visible light and/or near infra red light. 
     
     
         239 . The imaging composition of  claim 228 , wherein the first portion is joined to the common protein via a succinimide-amine linkage. 
     
     
         240 . The imaging composition of  claim 228 , wherein the second portion is joined to the common protein via a succinimide-amine linkage. 
     
     
         241 . The imaging composition of  claim 228 , wherein the first portion is joined to the common protein via a maleimide-thiol linkage. 
     
     
         242 . The imaging composition of  claim 228 , wherein the second portion is joined to the common protein via a maleimide-thiol linkage. 
     
     
         243 . The imaging composition of  claim 228 , wherein the first portion and/or the second portion is joined to the common protein via an iodoacetamide-thiol linkage. 
     
     
         244 . The imaging composition of  claim 228 , wherein the first portion and/or the second portion is joined to the common protein via an amine-carboxylic acid linkage. 
     
     
         245 . The imaging composition of  claim 228 , wherein the first portion and/or the second portion is joined to the common protein via a hydrazide-aldehyde linkage. 
     
     
         246 . The imaging composition of  claim 228 , where the first portion and/or the second portion is joined to the common protein via a hyrdazide-ketone linkage. 
     
     
         247 . A photoswitchable imaging composition, comprising:
 a solution comprising a reducing agent and a light-emitting molecular entity, the light-emitting molecular entity in the solution being capable of being switched between a first state able to emit light at a first, emission wavelength upon excitation and a second state that does not substantially emit light at the first wavelength upon excitation,   wherein the light-emitting entity comprises a first portion that is capable of emitting light at the first wavelength upon excitation when the light-emitting entity is in its first state, and a second portion that activates the first portion upon exposure to an external stimulus, thereby placing the light-emitting entity in its first state.